KR101578955B1 - Hydroxyalkyl starch derivatives and process for their preparation - Google Patents

Abstract

The present invention relates to a process for the preparation of hydroxyalkyl starch (HAS) through an optionally oxidized reducing end of such HAS, independently of the amino group, a specifically protected carbonyl group, i. E. An amino group of a crosslinking compound comprising an acetal group or a ketal group And reacting the hydroxyalkyl starch derivative with a base.

Description

[0001] Hydroxyalkyl starch derivatives and processes for their preparation [0002]

The present invention relates to a process for the preparation of hydroxyalkyl starch (HAS) through an optionally oxidized reducing end of such HAS, independently of the amino group, a specifically protected carbonyl group, i. E. An amino group of a crosslinking compound comprising an acetal group or a ketal group And reacting the hydroxyalkyl starch derivative with a base. This method may further comprise the step of reacting the thus obtained HAS derivative with the amino group of the biologically active compound via alkylation, preferably reductive amination. Furthermore, the present invention relates to a HAS derivative obtainable or obtained by the method of the present invention, and a specific HAS derivative per se. The present invention also relates to the use of specific HAS derivatives for the production of these HAS derivatives and drugs as pharmaceutical compositions, therapeutic agents or prophylactic agents comprising HAS derivatives containing biologically active compounds.

Hydroxyalkyl starch (HAS), especially hydroxyethyl starch (HES), is a substituted derivative of the naturally occurring carbohydrate polymer amylopectin, present in corn starch at a concentration of up to 95% by weight and degraded by alpha-amylase in the body. HES exhibits particularly favorable biological properties and is used as a blood volume substitute and is used in hemodilution therapy in special hospitals (Sommermeyer et al., 1987, Krankenhauspharmazie, 8 (8), 271-278; Weidler et al., 1991, Arzneimittel-forschung / Drug Res., 41, 494-498].

Several methods of making hydroxyethyl starch derivatives are described in the art.

DE 26 16 086 describes a method of conjugating hemoglobin with hydroxyethyl starch, wherein in the first step a cross-linking agent (e.g. bromocyan) is combined with hydroxyethyl starch and subsequently hemoglobin is reacted with the intermediate product .

One important area in which HES is used is to stabilize the polypeptide, for example, applied to the circulatory system, in order to obtain a particular physiological effect. One specific example of these polypeptides is erythropoietin, an approximately 34,000 kDa acid glycoprotein that is essential in regulating the level of red blood cells in circulation.

A problem known to occur when applying polypeptides and enzymes is that these proteins often exhibit unsatisfactory stability. In particular, erythropoietin has a relatively short plasma half-life (see Spivak and Hogans, 1989, Blood 73, 90; McMahon et al., 1990, Blood 76, 1718). This means that the therapeutic plasma level is rapidly lost and that intravenous administration must be repeatedly performed. Furthermore, under certain circumstances, an immune response against the peptide is observed.

In general, it is believed that the stability of such polypeptides can be improved when the polypeptides are coupled with polymeric molecules, and the immune response against these polypeptides is reduced.

WO 94/28024 discloses that physiologically active polypeptides modified with polyethylene glycol (PEG) exhibit reduced immunogenicity and antigenicity and are cyclic in the bloodstream for significantly longer periods of time than unconjugated proteins, . However, PEG-drug conjugates show some disadvantages, for example they do not exhibit the natural structure that can be recognized by in vivo degradation pathway elements. Thus, other conjugates and protein polymerizations distinct from PEG-conjugates have been generated.

WO 02/080979 discloses a compound comprising a conjugate of an active agent and a hydroxyalkyl starch, wherein the active agent and the hydroxyalkyl starch are linked either directly or through a linker compound. As far as direct linking is concerned, the reaction of the active agent with the hydroxyalkyl starch is carried out in an aqueous medium comprising at least 10% by weight of water. No examples of hydroxyalkyl starches connected with such crosslinkable compounds through the amino groups of the crosslinkable compounds further containing a protected carbonyl group were provided at all. In addition, no example was provided to indicate a HAS derivative obtained by reacting the HAS derivative via the carbonyl group with the amino group of the biologically active agent.

WO 03/074087 discloses that the binding between a hydroxyalkyl starch molecule and a protein is covalent and that the hydroxyalkyl starch, which is the result of coupling of the functional group generated from the reaction of the end aldehyde group of the hydroxyalkyl starch with the functional group of the protein and the aldehyde group Protein conjugates are described.

WO 03/074088 discloses that the linkage between a hydroxyalkyl starch and a low molecular weight compound is covalent and that the molecular weight of the hydroxyalkyl starch is lowered as a result of the coupling of the functional group generated from the reaction of the terminal aldehyde group of the hydroxyalkyl starch, A hydroxyalkyl starch conjugate with a compound is disclosed.

WO 2005/014024 discloses a polymer functionalized with an aminooxy group or derivative thereof, a conjugate in which the functionalized polymer is covalently coupled with a protein by an oxime linking group, a process for producing the functionalized polymer, A method of manufacture, a functionalized polymer obtainable by the method of the invention, a conjugate as obtainable by such method and a pharmaceutical composition comprising at least one conjugate, and the conjugate and composition for the prevention or treatment of the human or animal body Is described.

WO 2005/092390 describes conjugates of hydroxyalkyl starch and proteins formed by covalent linkage of derivatives of hydroxyalkyl starch or hydroxyalkyl starch with proteins, methods of making these conjugates, and the use of these conjugates have.

WO 2004/024777 describes hydroxyalkyl starch derivatives which can be obtained by a method of reacting hydroxyalkyl starch derivatives, particularly hydroxyalkyl starches, with primary or secondary amino groups of linker compounds. According to a particularly preferred embodiment, WO 2004/024777 discloses a process for the preparation of a compound of formula I in which a hydroxyalkyl starch is reacted with a primary or secondary amino group of a linker compound and the resulting reaction product is reacted with a polypeptide, , Particularly preferably erythropoietin, is described in JP-A-11-19455. A particularly preferred hydroxyalkyl starch is a hydroxyethyl starch. According to WO 2004/024777, a hydroxyalkyl starch, preferably a hydroxyethyl starch, is reacted with a linker compound at its reducing end which is not oxidized prior to the reaction.

WO 2004/024776 discloses a method of reacting a hydroxyalkyl starch derivative, particularly a hydroxyalkyl starch, with a primary or secondary amino group of a crosslinkable compound, or by reacting it with two crosslinkable compounds (a hydroxyalkyl The starch derivative has at least one functional group X capable of reacting with the functional group Y of the additional compound and the group Y of the further compound is a hydrolyzable group such as an aldehyde group, a keto group, a hemiacetal group, an acetal group or a thio group Roxy alkyl starch derivatives are described. According to a particularly preferred embodiment, WO 2004/024776 discloses a process for preparing hydroxyalkyl starch which comprises reacting a hydroxyalkyl starch with a primary or secondary amino group of a crosslinkable compound and optionally reacting the resulting reaction product with a second crosslinkable compound (The resulting hydroxyalkyl starch derivative has at least one functional group X capable of reacting with the functional group Y of the further compound, wherein the group Y is an aldehyde group, a keto group, a hemiacetal group, an acetal group or a thio group) Hydroxyalkyl < / RTI > obtained by a process comprising reacting the resulting reaction product with a polypeptide comprising at least one of said functional groups Y, preferably a polypeptide such as AT III, IFN-beta or erythropoietin, Starch derivatives. A particularly preferred hydroxyalkyl starch is a hydroxyethyl starch. According to WO 2004/024776, a hydroxyalkyl starch, preferably a hydroxyethyl starch, is reacted with a linker compound at its reducing end optionally oxidized prior to the reaction.

WO 2005/092928 describes conjugates of hydroxyalkyl starch, preferably hydroxyethyl starch and protein, which are conjugated to one or more of the aldehyde groups of the hydroxyalkyl starch or hydroxyalkyl starch derivative and one or more amino groups The hydroxyalkyl starch or derivative thereof is covalently linked to the protein via an azomethine chain or an aminomethylene chain. WO 2005/092928 also relates to methods of making these conjugates and their specific uses.

US 2006/0194940 A1 describes water soluble polymer alkanals. In particular, protected aldehyde reagents are described which react with the polymer. Although poly (saccharides) are generically mentioned, a particularly preferred polymer is polyethylene glycol. Starch, or particularly modified starches, such as hydroxyalkyl starches, are not described in US 2006/0194940 A1. As a result, US 2006/0194940 A1 does not describe a specific method for coupling a given linker compound with a hydroxyalkyl starch. The same is true for US 7,157,546 B2, EP 1 591 467 A1 and WO 2004/022630 A2.

US 6,916,962 B2 describes aminoacetal crosslinking compounds in unprotected and protected forms. No discussion of possible coupling of such cross-linking compounds with polymers other than polyethylene glycol is included in the document. In particular, modified starches such as hydroxyalkyl starches, as well as starches, are not described in US 6,916,962 B2. As a result, US 6,916,962 B2 does not contain any description of the specific manner in which the desired linker compound is coupled with the hydroxyalkyl starch. The same is true for US 6,956,135 B2 and WO 03/049699 A2.

US 5,990,237 describes structures containing protected aldehyde groups. The compound comprising these structures is preferably coupled with polyethylene glycol and this coupling is carried out through a halide as a functional group contained in the protected aldehyde group containing compound wherein the halide group reacts with the hydroxy group of the polyethylene glycol .

It is an object of the present invention to provide a novel method for obtaining a hydroxyalkyl starch derivative.

It is a further object of the present invention to provide a HAS derivative obtainable or obtainable by reacting a novel HAS derivative, for example HAS, with a specifically functionalized cross-linking compound.

It is a further object of the present invention to provide a novel novel HAS derivative, for example a HAS derivative, which is obtained or obtainable by reacting a HAS with a specifically functionalized cross-linking compound, with suitable functional groups of the biologically active compound To obtain a HAS derivative obtainable or obtainable by the reaction.

Surprisingly, for the preparation of specific HAS derivatives, one can be selectively coupled with an optionally oxidized reducing end of the hydroxyalkyl starch, on the one hand, through an amino group, and on the other hand a carbonyl group , That is, a crosslinkable compound having an acetal group or a ketal group can be used. Compared to the embodiment using a free aldehyde or keto group as the functional group, or a crosslinking compound having a hemiacetal group, for example, the use of such a fully protected group is advantageous over the reaction between the HAS and the crosslinking compound Thereby minimizing the risk of undesirable oligomerization or polymerization between the compound molecules. Unexpectedly, the acetal group and the acetal group contained in the resulting HAS derivative, which is characteristic of numerous functional groups such as an ether group, without at least partially disrupting the specific chemical structure of the hydroxyalkyl starch, especially the hydroxyethyl starch, Or deprotect the ketal group. Accordingly, the present invention is directed to providing a HAS derivative which is capable of effectively deprotecting the functional group of the resulting HAS derivative without at least partially destroying the HAS structure, in order to provide a HAS derivative with effective coupling with the biologically active compound, Taking account of the extremely effective method of making the first HAS derivative by minimizing the risk of oligomerization or polymerization between the binding compound molecules.

Therefore,

(i) reacting a hydroxyalkyl starch (HAS) of formula (I) with an amino group M of a cross-linking compound according to formula II via a carbon atom C * at the reducing end of the HAS, To obtain a derivative of the hydroxyalkyl starch derivative,

(I)

≪ RTI ID = 0.0 &

M-L-A

(III)

In this formula,

A is an acetal group or a ketal group; L is a spacer bridging M and A; C * is optionally oxidized prior to reaction with HAS and M;

X is a functional group generated by reacting the amino group M with HAS through the optionally oxidized reducing end carbon atom C * of HAS;

HAS 'is the remainder of the hydroxyalkyl starch molecule; R ₁ , R ₂ and R ₃ are independently hydrogen or a linear or branched hydroxyalkyl group.

In addition, the present invention relates to hydroxyalkyl starch (HAS) derivatives obtainable or obtainable by said process.

Furthermore, the present invention relates to hydroxyalkyl starch (HAS) derivatives of formula III:

(III)

In this formula,

A is an acetal or ketal group; L is a spacer bridging X and A;

X is selected from the group consisting of the amino group M of the crosslinking compound of the formula (II), the carbon atom C * of the hydroxyalkyl starch (HAS) of the following formula I, wherein C * is optionally oxidized before the reaction with HAS and M, Lt; RTI ID = 0.0 > HAS < / RTI >

HAS 'is the remainder of the hydroxyalkyl starch molecule; R ₁ , R ₂ and R ₃ are, independently, hydrogen or a linear or branched hydroxyalkyl group:

Formula I

(II)

M-L-A

Hydroxyalkyl  Starch

The term "hydroxyalkyl starch" (HAS) in the context of the present invention refers to a starch derivative substituted by one or more hydroxyalkyl groups. Preferred hydroxyalkyl starches of the present invention have the configuration according to formula I ':

(I ')

Wherein HAS 'is the remainder of the hydroxyalkyl starch molecule; R _1, R ₂ and R ₃ are independently hydrogen, a linear or branched hydroxyalkyl group, or a group ^{- [(CR 1 R 2)} m O] n [CR 3 R 4] o -OH ( wherein, R ¹ , R ² , R ³ and R ⁴ are independently selected from the group consisting of hydrogen and an alkyl group, preferably a hydrogen and a methyl group, m is from 2 to 4, and the residues R ¹ and R ² are m groups CR ¹ R ² , n is from 0 to 20, preferably from 0 to 4, o is from 2 to 20, preferably from 2 to 4, with the proviso that R ³ and R ⁴ in the o group CR ³ R ⁴ are the same Or may be different).

Preferably, R ₁ , R ₂ and R ₃ are independently a group - (CH ₂ CH ₂ O) _n -H, wherein n is an integer, preferably 0, 1, 2, 3, 4, And in particular R ₁ , R ₂ and R ₃ are independently hydrogen or 2-hydroxyethyl.

In formula (I) and (I '), the reducing end of the starch molecule is presented in a non-oxidized form, and the terminal saccharide unit of the HAS is in the form of a hemiacetal form which may exist in equilibrium with (free) Respectively. The abbreviation HAS 'as used in the context of the present invention refers to a HAS molecule that does not carry a terminal saccharide unit at the reducing end of the HAS molecule. As used herein in the context of the present invention means " the remainder of the hydroxyalkyl starch molecule ".

As used herein, the term "hydroxyalkyl starch" refers to a starch hydrolyzate wherein the terminal carbohydrate moiety comprises a hydroxyalkyl group R ₁ , R ₂ and / or R ₃ as shown for the sake of brevity in formulas I and I ' As well as at least one hydroxy group present anywhere in the terminal carbohydrate moiety and / or the remainder of the hydroxyalkyl starch molecule HAS 'is bound to the hydroxyalkyl group R ₁ , R ₂ and / or R ₃ Substituted < / RTI >

Hydroxyalkyl starch comprising two or more different hydroxyalkyl groups is also possible.

The one or more hydroxyalkyl groups contained within the HAS may contain one or more, especially two or more, hydroxy groups. According to a preferred embodiment, at least one hydroxyalkyl group contained in the HAS contains one hydroxy group.

The expression "hydroxyalkyl starch" also includes derivatives wherein the alkyl group is mono- or poly-substituted. In this context, it is preferred that the alkyl group be substituted with a halogen, especially a fluorine, or an aryl group. Furthermore, the hydroxy group of the hydroxyalkyl group may be esterified or etherified.

Further, instead of alkyl, a linear or branched substituted or unsubstituted alkenyl group may be used.

Hydroxyalkyl starch is an ether derivative of starch. In addition to these ether derivatives, other starch derivatives may be used in the context of the present invention. For example, derivatives containing esterified hydroxy groups are useful. These derivatives may be, for example, derivatives of unsubstituted mono- or dicarboxylic acids having 2 to 12 carbon atoms or substituted derivatives thereof. Particularly useful are derivatives of unsubstituted monocarboxylic acids having 2 to 6 carbon atoms, especially derivatives of acetic acid. In this context, acetyl starch, butyryl starch and propionyl starch are preferred.

Furthermore, derivatives of unsubstituted dicarboxylic acids having 2 to 6 carbon atoms are preferred.

In the case of derivatives of dicarboxylic acids, it is useful that the second carboxy group of such a dicarboxylic acid is also esterified. Furthermore, derivatives of monoalkyl esters of dicarboxylic acids are also suitable in the context of the present invention.

In the case of a substituted mono- or dicarboxylic acid, the substituent group may preferably be the same as mentioned above for the substituted alkyl residue.

Techniques for esterifying starch are known in the art (see, for example, Klemm D. et al, Comprehensive Cellulose Chemistry Vol. 2, 1998, Whiley-VCH, Weinheim, New York, especially chapter 4.4, Esterification of Cellulose (ISBN 3-527-29489-9).

According to a preferred embodiment of the present invention, a hydroxyalkyl starch according to the above-mentioned formula (I) is used. Other saccharide ring structures included within the HAS 'can be the same or different from the stated saccharide rings, provided that they lack a reducing end if different.

There are no particular restrictions on the moieties R ₁ , R ₂ and R ₃ according to formula I as far as they are concerned. According to a preferred embodiment, R ₁ , R ₂ and R ₃ are independently hydrogen, or a hydroxyalkyl group, a hydroxyaralkyl group or a hydroxyalkaryl group having 2 to 10 carbon atoms in each alkyl residue . Hydrogen, and hydroxyalkyl groups having 2 to 10 carbon atoms are preferred. More preferably, the hydroxyalkyl group has 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, even more preferably 2 to 3 carbon atoms. In a preferred embodiment, the hydroxyalkyl starch is a compound wherein R ₁ , R ₂ and R ₃ are independently hydrogen or a group (CH ₂ CH ₂ O) _n -H, wherein n is an integer, preferably 0, , 4, 5, or 6).

Thus, "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch, and hydroxybutyl starch, with hydroxyethyl starch and hydroxypropyl starch being particularly preferred, and hydroxyethyl starch being most preferred Do.

The alkyl, aralkyl and / or alkaryl groups may be linear or branched and may be suitably substituted.

Accordingly, the present invention also relates to HAS derivatives and methods as mentioned above wherein R ₁ , R ₂ and R ₃ are independently hydrogen, or a linear or branched hydroxyalkyl group having 2 to 6 carbon atoms .

Thus, R ₁ , R ₂ and R ₃ are preferably selected from the group consisting of H, hydroxyhexyl, hydroxypentyl, hydroxybutyl, hydroxypropyl, such as 2-hydroxypropyl, 3-hydroxypropyl, Hydroxyethyl, such as 2-hydroxyethyl, and hydrogen and 2-hydroxyethyl groups are particularly preferred.

Accordingly, the present invention also relates to HAS derivatives and methods as mentioned above wherein R ₁ , R ₂ and R ₃ are, independently, hydrogen or a 2-hydroxyethyl group, and one of R ₁ , R ₂ and R ₃ Is more preferably 2-hydroxyethyl.

Hydroxyethyl starch (HES) is most preferred for all aspects of the present invention.

Accordingly, the present invention relates to HAS derivatives and methods as mentioned above wherein the polymer is a hydroxyethyl starch and the derivative is a hydroxyethyl starch (HES) derivative.

HAS, especially HES, is mainly characterized by molecular weight distribution, degree of substitution and C ₂ : C ₆ substitution ratio. There are two possibilities to describe the degree of substitution:

The degree of substitution (DS) of HAS is a relative description of the glucose monomer moiety substituted on the basis of all glucose moieties.

The substitution pattern of HAS may also be described as molar substitution (MS), which is the number of hydroxyethyl groups per glucose moiety.

In the context of the present invention, the substitution pattern of HAS, preferably HES, is referred to as MS as mentioned above (Sommermeyer et al., 1987, Krankenhauspharmazie, 8 (8), 271-278, in particular p. 273].

MS is determined by gas chromatography after fully hydrolyzing the HES molecule. MS values of each HAS, especially HES starting material, are provided. This MS value is not affected at all during the derivatization step in steps a) and b) of the process of the invention.

HAS, especially the HES solution, is present as a polydisperse composition wherein each molecule is different from one another in terms of degree of polymerization, number of branching sites and pattern, and substitution pattern. Thus, HAS, especially HES, is a mixture of compounds with different molecular weights. As a result, the specific HAS, especially the HES solution, is determined by the average molecular weight with the aid of statistical means. In this context, M _n is calculated as an arithmetic average according to the number of molecules. On the other hand, the M _w (or MW) weight average molecular weight represents a unit which depends on the specific mass of the HAS, in particular HES.

In this context, the number average molecular weight is defined by the following equation:

Where n _i is the number of molecules of species i of the molar mass M _i ;

는 해당 값이 평균이라는 것을 표시하지만, 선이 통상적으로 생략된다.

Indicates that the value is an average, but the line is usually omitted.

M _w is the weight average molecular weight defined by the following equation:

Where n _i is the number of molecules of species i of the molar mass M _i ;

는 해당 값이 평균이라는 것을 표시하지만, 선이 통상적으로 생략된다.

Indicates that the value is an average, but the line is usually omitted.

Preferably, the hydroxyalkyl starch, particularly the hydroxyethyl starch, used in the present invention has an average molecular weight (weight average) of from 1 to about 1000 kDa, more preferably from about 1 to about 800 kDa, more preferably from about 1 to about 500 kDa. The hydroxyethyl starch may further have a preferred molar substitution of from 0.1 to 3, preferably from 0.1 to 2, more preferably from 0.1 to 0.9 or from 0.4 to 2, preferably from 0.4 to 1.3, C ₂ : C ₆ substitution ratio may be in the range of 2 to 20.

The term "average molecular weight" as used in the context of the present invention is described in Sommermeyer et al., 1987, Krankenhauspharmazie, 8 (8), 271-278; (Low Angle Laser Light Scattering) -GPC method as described in Weidler et al., 1991, Arzneim. -Forschung / Drug Res., 41, 494-498. In addition, if the average molecular weight is below 10 kDa, calibration was performed using a standard previously found to be suitable by LALLS-GPC.

According to a preferred embodiment of the present invention, the average molecular weight of the hydroxyethyl starch used is from about 1 to about 1000 kDa, more preferably from about 1 to about 800 kDa, more preferably from about 1 to 500 kDa, About 400 kDa, more preferably about 5 to about 300 kDa, more preferably about 10 to about 200 kDa, especially about 50 to about 150 kDa.

Further, the molar substitution of HAS, particularly HES, is preferably carried out at a concentration of from about 0.1 to about 3, preferably from about 0.4 to about 1.3, such as 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, to be.

One example of a HES having an average molecular weight of about 5 to 300 kDa, preferably 50 to 150 kDa is a HES having a molar substitution of 0.1 to 3, preferably 0.4 to 1.3, such as 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 , 1.1, 1.2, or 1.3.

As far as the C ₂ : C ₆ substitution ratio is concerned, such a substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, even more preferably in the range of 3 to 12.

Starch other than hydroxyalkyl starch

Generally, the method of the present invention can also be carried out, and the derivatives of the present invention can also be prepared using hydroxyalkyl starches as mentioned above, particularly starches other than hydroxyethyl starches, Must also contain a reducing end in the form of a hemiacetal, optionally present in equilibrium with the (free) aldehyde form, which may be oxidized to provide the respective oxidized form. In particular, highly branched, unmodified or less substituted starch products, i.e., those having a degree of branching significantly higher than amylopectin, having an alpha-1, 6-branching degree of glycogen or even exceeding that, Of the starch is 0.3 or less, preferably 0.05 to 0.3. The term MS (molar substitution) as used in the context of such highly branched, unsubstituted or low substituted starch products means the average number of hydroxyethyl or hydroxypropyl groups per anhydroglucose unit. MS is typically determined by determining the amount of hydroxyethyl or hydroxypropyl groups in the sample and the amount of computerized quota for the anhydroglucose unit present therein. The MS may be determined by gas chromatography. The degree of branching can be determined by gas chromatographic methylation analysis as mole% of anhydrous glucose linked in alpha-1,4,6-glycosidic in the polymer. The degree of branching is an average in all cases because the highly branched, unmodified or low substituted starch product of the present invention is a polydisperse compound. The glucose units in the highly branched, unsubstituted or low substituted starch products are linked via alpha-1,4- and alpha-1,6-chains. The degree of branching refers to the ratio of alpha-1,4,6-linked glucose units as mole% based on all anhydrous glucose. The C ₂ / C ₆ ratio represents the substitution at C-2 versus the substitution ratio at C-6. Highly branched, unsubstituted or low substituted starch products have a preferred degree of branching of 6% to 50%, which can be achieved by the glucose transfer step with the aid of a branching enzyme. Even more preferably, the degree of branching is in the range of 10 to 45, more preferably in the range of 20 to 40, for example 20, 25, 30, 35, or 40. Further, it is preferably in the range of more than 20 to 40, preferably in the range of more than 20 to 30, for example, 21 to 40, preferably 21 to 30. The starting materials that can be used for this purpose are all starches in principle, but waxy starches with a high amylopectin ratio or amylopectin fraction are preferred. The degree of branching of the starch product (so long as these "other starches" are involved) required for use according to the present invention is in the range of 8% to 20%, expressed as mole% of anhydrous glucose. This means that the starch product that can be used for the purposes of the present invention has an average of one alpha-1, 6-chain, so that it has a fraction of 12.5 to 5 glucose units per minute. The preferred highly branched, unsubstituted or low substituted starch products have a degree of branching of greater than 10% to less than 20%, especially 11 to 18%. The higher degree of branching means that the solubility of the starch products of the present invention is greater and the bioavailability of these dissolved starch products in the body is greater. Particularly preferred is an unmodified starch product with a degree of branching of more than 10%, in particular 11% to 18%. Highly branched, unsubstituted or low substituted starch products can be prepared by partially derivatizing a free hydroxyl group with a hydroxyethyl or hydroxypropyl group, optionally followed by a targeting enzymatic assembly using a so-called branching or transferase have. Instead, it is possible to convert hydroxyethylated or hydroxypropylated starches into highly branched, unsubstituted or low substituted starch products by enzymatic assembly using so-called branching or transferases. The obtaining of enzymatically branched starch products from wheat starch with a degree of branching of less than 10% is known per se and is described, for example, in WO 00/66633 A. Suitable branching or transferases and their methods of obtaining are described in WO 00/18893 A, US 4,454,161, EP 0 418 945 A, JP 2001294601 A or US 2002/065410 A. These latter disclosures disclose unmodified starch products with a degree of branching of more than 4% to 10% or more. Enzymatic Glycosylation This reaction can be carried out in a manner known per se, for example waxy corn starch; Potato starch obtained from a potato having a high amylopectin content; Or corn with a high content of amylopectin, corn with a high amylopectin content, or corn with a high amylose content, in an aqueous solution at a pH of from 6 to 8 and at a temperature of from 25 to 40 DEG C Followed by incubation with appropriate enzymes under mild conditions. The molecular weight M _w as used in the context of highly branched, unsubstituted or low substituted starch products means weight average molecular weight. This can be determined in a manner known per se by various methods, namely gel permeation chromatography (GPC) or high performance liquid chromatography (HPLC) in conjunction with light scattering and RI detection. The preferred C ₂ / C ₆ ratio for substituted starches ranges from 5 to 9. The high degree of branching of the highly branched, unmodified or low substituted starch product is dependent on the water solubility of the starch product to such an extent that it completely or substantially eliminates the hydroxyethyl or hydroxypropyl substitution to maintain such starch product in solution . The average molecular weight of highly branched, unsubstituted or low substituted starch products can be increased in a suitable manner through the permeability limitation of the peritoneum. A characteristic parameter that can be used in this case is also the GPC value of the so-called bottom fraction BF 90% (molecular weight at 90% peak area as a measure of the fraction of lesser molecular fractions). Larger ultrafiltration (UF) efficiency can be achieved by moderately increasing the molecular weight while simultaneously reducing the absorption rate across the peritoneum. At the same time, the high molecular weight residual fragments produced by degradation by endogenous amylase, which can no longer be further degraded by the amylase and which are stored in organs or tissues, are no longer present or only to a small extent.

According to the invention, the hydroxyalkyl starch is reacted with a crosslinkable compound M-L-A, wherein M is an amino group, A is an acetal group or a ketal group, and the groups M and L are separated by suitable spacers.

Acetal or ketal group A

As long as the acetal group or the ketal group A is concerned, there is no specific limitation thereto. In the context of the present invention, the term "acetal group" also includes sulfur acetals and nitrogen acetals, and the term "ketal group" also includes sulfur ketals and nitrogen ketals. Additionally, so long as the term "acetal group" is concerned, so long as the hemiacetal is expressly excluded and the term "ketal group" is concerned, the hemi ketal is expressly excluded.

According to a preferred embodiment of the present invention, the group A of the crosslinking compound M-L-A is a residue according to the following formula IIa:

≪ RTI ID = 0.0 &

In this formula,

Z ₁ and Z ₂ are each independently O or S or NR _x , preferably O and R _x is H or lower alkyl such as methyl, ethyl or propyl, such as n- or i-propyl, or C (O) -R _y (where, R _y is preferably, C ₁ -C ₆ alkyl and C ₆ is selected from the group consisting of -C ₁₄ aryl, and more preferably, is not optionally substituted and preferably substituted, Is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl; R _x is preferably H;

A ₁ and A ₂ are each independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, benzyl, 1,1,1- trichloroethyl, nitrobenzyl, methoxybenzyl Lt; / RTI > benzyl, or forms a ring according to the following formula < RTI ID =

≪ RTI ID =

In this formula,

A ₁ and A ₂ together are - (CH ₂ ) ₂ - or - (CH ₂ ) ₃ - or - (CH ₂ CH (CH ₃ )) - and A ₃ is H or methyl, ethyl, Isopropyl, n-butyl, isobutyl, pentyl, benzyl, or together with the N atom of the amino group M or together with the appropriate atoms contained in L form a ring.

Preferably, at least one of Z ₁ and Z ₂ is O, more preferably both Z ₁ and Z _{2 are} O.

As far as residue A ₃ is concerned, an acetal group is preferred according to the invention, i.e. A ₃ is preferably H.

또는

또는

또는

When A is a ketal group, it is preferable that A < ₃ > is methyl. Thus, the metal group A which can be considered according to the invention is, in particular,

or

or

or

또는

이다.or

or

to be.

A ₃ , for example, together with the N atom of the amino group M or together with the appropriate atoms contained in L, form a ring, the crosslinkable compounds contemplated according to the invention are, for example:

또는

또는

or

or

Particularly preferred crosslinking compounds according to the invention are

인데, 즉 아미노 기 M이 2급 아민이고, Z₁ 및 Z₂ 둘 다가 O이며, A₁ 및 A₂가 함께, -(CH₂)₂-이다.

That is, the amino group M is a secondary amine, both of Z ₁ and Z _{2 are} O, and A ₁ and A ₂ together form - (CH ₂ ) ₂ -.

According to a preferred embodiment, A ₁ and A ₂ are each methyl or ethyl, more preferably ethyl. Thus, a particularly preferred acetal group A is -CH (OCH _{3) 2} or -CH (OC ₂ H _{5) 2,} in particular -CH (OC ₂ H _{5) 2} according to the invention.

및

이다.According to a further embodiment of the A ₁ and A ₂ form a ring according to formula IIb, A ₁ and A ₂ together is preferably - (CH _{2) 2} - a. As far as this aspect is concerned, the particularly preferred acetal group A according to the invention is

And

to be.

Amino group M

As far as the amino group M is concerned, there is no particular limitation, except that the amino group can be oxidized or reacted with a non-oxidized reducing end, i.e. in a non-oxidised state (i.e. as a hemiacetal or free aldehyde group) Or in the oxidized state (i.e. as a lactone or free carboxyl group), through the carbon atom C * of the HAS, preferably the reducing end saccharide unit of the HES. The term "amino group ", as used in the context of this present application, also includes amino groups such as, for example, a protonated amino group and a pharmaceutically acceptable anion such as a chloride, hydrogen sulphate, sulfate, carbonate, hydrogen carbonate, Phosphate, or a suitable salt of hydrogen phosphate.

Preferably, the amino group of the crosslinking compound M-L-A according to the invention is a group according to the following formula IIc:

(IIc)

으로 이루어진 군 중에서 선택된 화학적 부분이며;Wherein Y is absent or

A chemical moiety selected from the group consisting of:

Wherein G is O or S or NH, G is preferably O, < RTI ID = 0.0 >

R 'is H or a hydroxy group or an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, aralkyl, substituted aralkyl, Alkylaryl, substituted alkylaryl, and the like. In this context, the term "alkyl" relates to unbranched alkyl residues, branched alkyl residues and cycloalkyl residues. Preferably, each of these organic residues has from 1 to 10 carbon atoms. As contemplated substituents, halogens such as F, Cl or Br may be mentioned. Preferably, the organic moiety is an unsubstituted hydrocarbon.

When R 'is a hydroxy group, the preferred amino group of the present invention is HO-NH-, that is, Y is absent.

Preferably, when R 'is an organic residue, R' is selected from the group consisting of alkyl and substituted alkyl, with alkyl residues being particularly preferred. Even more preferably, the optionally substituted alkyl moiety has 1 to 10, more preferably 1 to 6, more preferably 1 to 4, such as 1, 2, 3, or 4 carbon atoms. Thus, preferred organic moieties according to the invention are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl. According to a particularly preferred embodiment, the organic residue R 'is methyl or ethyl, in particular methyl.

Thus, when R 'is an organic moiety, preferred amino groups according to the invention are, for example, H ₃ C-CH ₂ -NH-, H ₃ C-NH-, H ₅ C ₆ -NH-, H ₃ C- _{CH 2 -NH-O-, H 3} C-NH-O-, H 5 C 6 -NH-O- , _{and, H 3 C-NH-, H} 5 C 6 -NH-, and H ₃ C-NH- O- is particularly preferred.

또는

또는

이다.
In accordance with the present invention, R 'is not a separate residue, to form a ring structure with residue A ₃ of group A in combination with compound or cross-linking with a suitable atom included in L is also possible. These structures are also included in the definition of the term "alkyl" mentioned above in connection with R '. For example, R 'may form a cyclic structure together with the residue A ₃ of the group A of the cross-linkable compound, where A is a ketal group. Conceivable crosslinking compounds are, for example,

or

or

to be.

인데, 즉 아미노 기 M이 2급 아민이고, Z₁ 및 Z₂ 둘 다가 O이며, A₁ 및 A₂가 함께, -(CH₂)₂-이다.In this case, a particularly preferred crosslinking compound according to the present invention is

That is, the amino group M is a secondary amine, both of Z ₁ and Z _{2 are} O, and A ₁ and A ₂ together form - (CH ₂ ) ₂ -.

In a preferred embodiment of the present invention, R 'is H. Therefore, the preferred amino group M of the present invention is

Wherein G is O or S, and when present two times, it is independently O or S and O is preferred.

When R 'is H, particularly preferred amino groups M of the present invention are H ₂ N-, H ₂ NO-, and H ₂ N-NH- (C═O) -.

Thus, the present invention also, the amino group M is _{H 2 N-, H 2 NO-,} H 2 N-NH- (C = O) -, H 3 C-NH- or H ₃ C-NH-O-, Preferably H ₂ N-, H ₂ NO-, or H ₂ N-NH- (C = O) -.

Spacer L

According to the present invention, the functional groups M and A of the crosslinkable compound are separated by suitable spacers. The term "spacer " as used in this context relates to all suitable chemical moieties bridging M and A.

In general, there is no particular limitation with regard to the chemical nature of the spacer L, except that L makes it possible to carry out the process according to the invention for the preparation of novel derivatives and, as far as their intended use is concerned, Which has a special chemical property.

According to a preferred embodiment of the present invention, L bridging M and A is a spacer comprising at least one structural unit according to the following formula IId:

≪ RTI ID = 0.0 &

In this formula,

L ₁ and L ₂ are, independently of each other, H or an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkyl Quot; is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted Aryl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl.

In this context, the term "alkyl" relates to unbranched alkyl residues, branched alkyl residues and cycloalkyl residues. As a preferred substituent, a halogen such as F, Cl or Br may be mentioned.

Preferably, L ₁ and L ₂ are independently of each other H or an organic residue selected from the group consisting of alkyl and substituted alkyl; More preferably, L < ₁ > and L < ₂ > are independently from each other H or alkyl; Even more preferably, both L < _{1 >} and L < ₂ >

Preferably, when L ₁ and L ₂ are organic residues, each of L ₁ and L ₂ is, independently, 1 to 20, preferably 1 to 10, more preferably 1 to 8, More preferably 1 to 4 carbon atoms. Particularly preferred are residues L ₁ and L ₂ such as optionally substituted, preferably unsubstituted, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary-butyl residues. According to the present invention, L < ₁ > may be H and L < ₂ > may be an organic residue as defined above.

As far as the integer n is concerned, n is preferably 1 to 20, preferably 1 to 10, more preferably 1 to 6, more preferably 1 to 4,

When the integer n exceeds 1, the groups (CL ₁ L ₂ ) may be the same or different from each other. According to a preferred embodiment of the present invention, the groups CL ₁ L ₂ directly connected to each other have the same configuration.

According to a preferred embodiment of the present invention, the spacer L comprises a structural unit according to formula IId, wherein L ₁ and L ₂ are as defined above. More preferably, the integer n is 1 to 20, more preferably 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. More preferably, each of the groups CL ₁ L ₂ is (CH ₂ ), so that the spacer L bridging M and A has the following structure:

N is an integer of 1 to 20, more preferably 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. More preferably, n is in the range of 1 to 6, more preferably in the range of 1 to 4, for example 1, 2, 3, or 4, especially 2.

Thus, according to a particularly preferred embodiment of the invention, spacer L is -CH ₂ -CH ₂ - a.

According to a further embodiment of the invention, the spacer L of the crosslinkable compound comprises at least one structural unit according to formula IId.

≪ RTI ID =

Wherein n, L < ₁ > and L < ₂ > are as defined above,

이며;

;

L further comprises at least one chemical moiety different from - (CL ₁ L ₂ ) -.

In this context, it may be mentioned that the spacer L consists of a further chemical moiety T which separates the structural units - (CL ₁ L ₂ ) _n - and - (CL ₁ L ₂ ) _n - from the groups M or A have. Also, an embodiment in which the spacer L comprises a structural unit - (CL ₁ L ₂ ) _n - and two further chemical moieties T ₁ and T ₂ , wherein T ₁ is - (CL ₁ L ₂ ) _n - , And T ₂ separates - (CL ₁ L ₂ ) _n - from the group A). Accordingly, the present invention also encompasses embodiments wherein the crosslinkable compound has one of the following structural formulas:

With respect to the chemical moieties T, T ₁ or T ₂ , there is no particular restriction as to their chemical properties, except that L makes it possible to carry out the process according to the invention for the preparation of new derivatives, It has special chemical properties that provide suitable chemical properties for new derivatives.

Thus, T, T ₁ and / or T ₂ may comprise an optionally substituted aryl moiety, a suitable heteroatom, a suitable functional group, and the like. As far as the functional groups are concerned, mention may be made of an embodiment wherein these functional groups result from the production of a crosslinkable compound which reacts at least a first compound and at least a second compound with each other to give a compound MLA. For example, a crosslinking compound MLA can be obtained by reacting a first compound M-L'-W ₁ and a second compound W ₂ -L "-A, wherein -L- is -L'-FL" - a, F represents the functional group resulting from the reaction of functional group W ₁ with functional group W _2, L 'and L "at least one of the structural unit _{- (CL 1 L 2) n} -. include such functional groups W ₁ and W ₂ may be suitably selected. for example, the group one of W ₁ and W _2, i.e. W ₁ or W ₂ may be selected from the group consisting of a functional group according to the following list, or other group, W ₂ or W ₁ is suitably selected and may form a chemical chain with W ₁ or W ₂ , wherein W ₂ or W ₁ is also preferably selected from among the above-mentioned groups:

A C-C-double bond or a C-C-triple bond or an aromatic C-C- bond;

A thio group or a hydroxy group;

-Alkylsulfonic acid hydrazide, arylsulfonic acid hydrazide;

- 1,2-diol;

- 1,2-amino-thioalcohols;

- azide;

- 1,2-aminoalcohol;

A derivative of an amino group containing an amino group -NH ₂ or a structural unit -NH-, for example, an aminoalkyl group, an aminoaryl group, an aminoaralkyl group or an alkarylamino group;

-Hydroxylamino group -O-NH ₂ , or a derivative of a hydroxylamino group containing a structural unit -O-NH-, such as a hydroxylalkylamino group, a hydroxylarylamino group, a hydroxylaralkylamino group Or a hydroxyl alkaryl amino group;

An alkoxyamino group, an aryloxyamino group, an aralkyloxyamino group, or an alkaryloxyamino group each containing a structural unit -NH-O-;

(= G) -M 'wherein G is O or S, and M' is a leaving group having a carbonyl group, for example,

   -OH or -SH;

   An alkoxy group, an aryloxy group, an aralkyloxy group or an alkaryloxy group;

   An alkylthio group, an arylthio group, an aralkylthio group or an alkarylthio group;

   An alkylcarbonyloxy group, an arylcarbonyloxy group, an aralkylcarbonyloxy group, an alkarylcarbonyloxy group;

   - an activated ester, for example an ester of a hydroxylamine having an imide structure (e.g. N-hydroxysuccinimide);

- -NH-NH _2, or -NH-NH-;

-NO ₂ ;

- a nitrile group;

A carbonyl group such as an aldehyde group or a keto group;

- carboxy group;

- a -N = C = O group or an -N = C = S group;

- vinyl halide groups such as vinyl iodide or vinyl bromide groups or triflates;

-C≡C-H;

_{- - (C = NH 2 Cl} ) -O -alkyl;

- group _{- (C = O) -CH 2} -Hal ( wherein, Hal is Cl, Br, or I);

_{- -CH = CH-SO 2 -} ;

A disulfide group comprising a structure -S-S-;

; - machine

;

. - machine

.

For example, W ₁ or W ₂ may be a carboxy group or an activated ester, and W ₂ or W ₁ may be an amino group or a hydroxy group. Therefore, F representing functional groups generated from the reaction of functional groups W ₁ and W ₂ Is an amide or an ester.

Thus, for example, a crosslinkable compound having a spacer L comprising a functional group other than the structural unit - (CL ₁ L ₂ ) _n - may have the following structure:

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

In the above, L 'and L "may or may not contain the structural unit - (CL ₁ L ₂ ) _n -.

Among these structures, the crosslinkable compound preferably has the following structure:

또는

or

Of these structures, when L 'and L "are present, a spacer in which they contain a structural unit - (CL ₁ L ₂ ) _n - is preferable. In this case, n is preferably in the range of 1 to 4 More preferably from 1 to 3, for example 1, 2 or 3. When a given spacer contains, for example, two structural units - (CL ₁ L ₂ ) _n -, each structure The exponent n of the unit may be the same or different.

Thus, it is preferred that the following crosslinkable compounds have the following configuration:

을 갖는다.In the above, each n is independently in the range of preferably 1 to 4, more preferably 1 to 3, for example 1, 2 or 3. Therefore, the preferred spacer L

Respectively.

Thus, particularly preferred crosslinking compounds containing - (C = O) -NH- are:

또는

or

또는

or

또는

or

More preferred crosslinkable compounds containing - (C = O) -NH- are:

또는

or

Even more preferably, both L < _{1 >} and L < ₂ > Thus, cross-linking compounds having the following configuration are particularly preferred:

또는

or

또는

or

또는

or

Most preferred are crosslinkable compounds having the following structure:

또는

or

For example, preferred cross-linking compounds of the present invention are as follows:

또는

또는

or

or

또는

또는

or

or

Again, to illustrate the structure discussed above, a cross-linkable compound that can be considered in the context of the present invention is

 Lt; / RTI >

According to a further embodiment of the present invention, the spacer L may comprise more than one structural unit - (CL ₁ L ₂ ) _n - and these structural units may be the same or different, / Or in terms of L < _{1 >} and L < ₂ >, and two or more of such structural units may be separated by a heteroatom such as O or S. Preferably, according to this embodiment, the spacer L is at least one structure unit _{- (CL 1 L 2) n1} -O- (CL 1 L 2) n2 -, preferably _{- (CH 2) n1 -O- (} CH 2 ) _n2 - (wherein, n1 is included the same or are different), and n2, and the spacer L is _{- (CL 1 L 2) n1} - are connected and cross-linking the amino group M of the binding compound through, i.e. the crosslinking compound Includes the following sub-structures: < RTI ID = 0.0 >

_{M- (CL 1 L 2) n1} -O- (CL 1 L 2) n2 -.

According to a preferred embodiment imaginable, a spacer structure can be mentioned, for example - ((CL ₁ L ₂ ) _n1 -O) _m - (CL ₁ L ₂ ) _n2 - where m is from 1 to 20, 1 to 10, more preferably 1 to 6, such as 1, 2, 3, 4, 5, or 6. Particularly preferably, m is 1, 2 or 3, more preferably 2 or 3. Preferably, n1 is 2 to 4, more preferably 2. Preferably, n2 is 1 to 4, more preferably 1 or 2. Thus, preferred structures are, for example, _{- ((CL 1 L 2)} 2 -O) m - (CL 1 L 2) -, and more preferably is _{- ((CH 2) 2 -O} ) m - (CH ₂ ) -.

Again, to exemplify the structure discussed above, the crosslinking compound preferably used in the context of the present invention is

또는

또는

이다.

or

or

to be.

According to a further embodiment of the invention, the groups M and A may be separated by two suitable chemical moieties, and at least one of them contains - (CL ₁ L ₂ ) _n -, so that the N atom of the group M And the C atom of ketal group A form a ring. Preferred embodiments have already been given above and have the following structure:

Particularly preferred crosslinking compounds of the invention are those having the group H ₂ N- or the group H ₂ NO- or the group H ₂ N-NH- (C═O) -, particularly preferably the group H ₂ N-, as the group M, Is a compound having an acetal group as the group A, preferably an acetal group having the following structure:

More preferably, Z ₁ and Z ₂ are O, more preferably A ₁ and A ₂ are both ethyl. More preferably, the spacer L comprises the structural unit - (CL ₁ L ₂ ) _n -, wherein L ₁ and L ₂ are preferably H and the integer n is more preferably 1 to 20, , More preferably 1 to 6, more preferably 1 to 4, and still more preferably 2.

Thus, preferred cross-linking compounds according to the invention are, for example:

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

or

또는

또는

or

or

또는

or

또는

or

Even more preferably, the crosslinkable compound is selected from the group consisting of structures (a2), (a4), (a11), (a12), (a14), (a16), and (a18). More preferably, the crosslinkable compound is selected from the group consisting of structures (a2), (a11), (a12), (a14), (a16), and (a18). In particular, the crosslinkable compound is selected from the group consisting of structures (a2), (a11), (a12), and (a16).

이다.Particularly preferred as crosslinking compound MLA is 1-amino-3,3-diethoxypropane

to be.

For example, the amino-acetal crosslinkable compounds which may be contemplated according to the present invention are:

 For example, amino-ketal crosslinkable compounds which may be contemplated according to the invention are:

Hydroxyalkyl  Starch derivative

Accordingly, the present invention relates to a hydroxyalkyl starch (HAS) derivative obtainable or obtained by the above-mentioned method.

Furthermore, the present invention relates to HAS derivatives of formula III:

(III)

In this formula,

A is an acetal or ketal group; L is a spacer bridging X and A;

X is selected from the amino group M of the crosslinking compound of formula II via a carbon atom C * of the hydroxyalkyl starch (HAS) of formula I, wherein C * is optionally oxidized prior to the reaction with HAS and M A functional group generated by reacting with the HAS;

HAS 'is the remainder of the hydroxyalkyl starch molecule; R ₁ , R ₂ and R ₃ are, independently, hydrogen or a linear or branched hydroxyalkyl group:

Formula I

(II)

M-L-A.

As far as the preferred embodiments relating to HAS, preferably HES, L, A, R ₁ , R ₂ and R ₃ are concerned, reference is specifically made to the embodiments mentioned above.

In addition, the present invention R _1, R ₂ and R ₃ are independently a group _{- (CH 2 CH 2 O)} n -H ( wherein, n is an integer, preferably 0, 1, 2, 3, 4, 5, Or 6). ≪ / RTI >

In addition, the present invention relates to a HAS derivative as mentioned above, wherein the hydroxyalkyl starch is hydroxyethyl starch (HES).

In addition, the present invention relates to a HAS derivative as mentioned above, wherein A is a residue according to formula < RTI ID = 0.0 > (IIa)

≪ RTI ID =

In this formula,

Z ₁ and Z ₂ are each independently O or S or NR _x , preferably O; R _x is H or lower alkyl, such as methyl, ethyl or propyl, preferably H;

A ₁ and A ₂ are each independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, benzyl, 1,1,1- trichloroethyl, nitrobenzyl, methoxybenzyl Lt; / RTI > benzyl, or forms a ring according to the following formula < RTI ID =

≪ RTI ID =

In this formula,

A ₁ and A ₂ together are - (CH ₂ ) ₂ - or - (CH ₂ ) ₃ - or - (CH ₂ CH (CH ₃ )) - and A ₃ is H or methyl, ethyl, Isopropyl, n-butyl, isobutyl, pentyl, benzyl, or together with the N atom of the amino group M or together with the appropriate atom contained in L, A ₃ is preferably H.

The exact chemical nature of group X of the HAS derivative according to the invention depends on the respective chemical nature of the group M, the oxidation state of the carbon atom C * at the reducing end of the HAS, and the reaction conditions used for the reaction, . According to an aspect of the present invention in which the carbon atom C * is used in an oxidized state and in a non-oxidized state, specific and preferable examples are discussed in detail below.

Preferably, as far as X is concerned, the present invention provides a compound of formula (I) wherein X is -CH = N-, -CH ₂ -NH-, -CH = NO-, -CH ₂ -NH-O-, -C -, -C (= O) -NH-NH-, -CH = N-NH- (C = O) - or -CH ₂ -NH-NH- (C = O) -, ₂ -NH-, -CH = N-, -CH = NO-, -CH ₂ -NH-O-, -CH = N-NH- (C = O) - and -CH ₂ -NH-NH- C═O) -, more preferably selected from the group consisting of -CH ₂ -NH-, -CH═N-, -CH═NO-, and -CH ₂ -NH-O-. Gt; HAS < / RTI >

For certain embodiments of group X, the terminal saccharide unit of the HAS as present in the HAS derivative is in a ring structure which may be in equilibrium with the open structure according to formula III above, Distribution, and so on. In these cases and for the purposes of the present invention, Formula III as provided above includes ring structures as well as open structures, and Formula III does not limit HAS derivatives to open structures only. Where specific and preferred examples are discussed in detail below, ring structures are presented in some instances.

Further, the present invention relates to a HAS derivative as mentioned above, wherein L bridging M and A is a spacer comprising at least one structural unit according to formula (IId), preferably a structural unit according to formula (IId) will be:

≪ RTI ID =

In this formula,

L ₁ and L ₂ are, independently of each other, H or an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkyl Aryl, substituted alkylaryl and moiety -OR ", wherein R" is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, Aryl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl; Is preferably H or an organic moiety selected from the group consisting of alkyl and substituted alkyl; More preferably H or alkyl; More preferably H;

n is an integer of 1 to 20, preferably 1 to 10, more preferably 1 to 6, more preferably 1 to 4,

HAS , Preferably HES Lt; RTI ID = 0.0 >

According to the present invention, HAS, preferably HES, can be reacted with the amino group M of the cross-linking compound via the carbon atom C * of the reducing end of such starch, wherein C * Lt; / RTI >

As used in the context of the present invention, "reacting HAS through a reducing end" or "reacting HAS through a reducing end carbon atom C *" means that the HAS is reacted primarily with (optionally selectively oxidized) Lt; / RTI > through a reducing end.

Thus, "reacting primarily through its (optionally selectively oxidized) reducing end" statistically means that more than 50%, preferably 55% or more, more preferably 60% or more of the HAS molecules used for a given reaction, , More preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% (For example, optionally 95%, 96%, 97%, 98% or 99%) of HAS molecules via at least one (optionally selectively oxidized) reducing end per HAS molecule, May be reacted in the same predetermined reaction via one or more additional suitable functionalities that are included in the polymer molecule and are not at a reducing end . One or more HAS molecule (s) may be reacted via one or more (optionally selectively oxidized) reducing end, and at the same time, one or more additional suitable functionalities that are not included in the reducing end (optionally optionally oxidized) , More preferably more than 55%, more preferably more than 60%, more preferably more than 50%, more preferably more than 60%, more preferably more than 60%, more preferably more than 60% , More preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% , For example 95%, 96%, 97%, 98% or 99% (optionally oxidized).

The term "reducing end " as used in the context of the present invention relates to the terminal aldehyde group of the HAS molecule which may exist as an aldehyde group and / or exist as a corresponding hemiacetal and / or acetal group, residue -OR ₃ according to the have the following structure that can exist when the -0-CH ₂ -CH ₂ -OH:

When the reducing end is oxidized, the oxidized reducing end is in the form of a carboxy group and / or the corresponding lactone.

Oxidized reducing end

Thus, according to a first aspect of the present invention, the cross-linking compound is reacted with an oxidized C * atom of the reducing end of the HAS, preferably HES, via an amino group.

Although oxidation can be carried out according to all suitable method (s) for producing the oxidized reducing end of the hydroxyalkyl starch, for example, see Sommermeyer et al., US 6,083,909, column 5, lines 63- 67, and column 7, lines 25-39; column 8, line 53 to column 9, line 20; Each of which is incorporated herein by reference in its entirety).

When HAS, preferably HES, is selectively oxidized, the lactone

And / or carboxylic acid

Or a suitable salt of a carboxylic acid, for example an alkali metal salt, preferably a sodium and / or potassium salt, is calculated, and HAS 'is preferably HES'.

According to a first alternative of the invention, this type of HAS, preferably HES, is reacted with the amino group M of the crosslinking compound itself.

According to a second alternative of the invention, the selectively oxidized HAS, preferably HES, at its reducing end is first reacted with a suitable compound to obtain a HAS, preferably HES, comprising a reactive carboxy group.

The introduction of the reactive carboxy group into the HAS, which is selectively oxidized at its reducing end, can be carried out by any compound suitable for all possible methods.

According to a specific method of the present invention, the HAS selectively oxidized at its reducing end is reacted at the oxidized reducing end with one or more alcohols, preferably one or more acidic alcohols, for example, a pK _A value of 6-12 Or an acidic alcohol in the range of 7 to 11. The molecular weight of such acidic alcohols may range from 80 to 500 g / mol, for example from 90 to 300 g / mol or from 100 to 200 g / mol.

Suitable acidic alcohols have acidic protons and are reacted with oxidized HAS to form each reactive HAS ester, preferably a reactive HAS ester

More preferably, the reactive HAS esters according to the formula

Lt; _{RTI ID = 0.0} > HOR _A < / RTI >

Preferred alcohols are N-hydroxysuccinimides such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, suitably substituted phenols such as p-nitrophenol, o, p- Dinitrophenol, o, o'-dinitrophenol, trichlorophenol, such as 2,4,6-trichlorophenol or 2,4,5-trichlorophenol, trifluorophenol, such as 2, 4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazole, such as hydroxybenzotriazole. Especially preferred is N-hydroxysuccinimide, particularly preferred are N-hydroxysuccinimide and sulfo-N-hydroxysuccinimide. All alcohols may be used alone or may be used as two or more suitable combinations thereof. In the context of the present invention, it is also possible to use compounds which release the respective alcohols, for example by adding a diester of a carboxylic acid.

Thus, the present invention also relates to a process for the preparation of HAS which is selectively oxidized at its reducing end by reacting such oxidized HAS with an acidic alcohol, preferably N-hydroxysuccinimide and / or sulfo-N-hydroxysuccinimide As described above. ≪ / RTI >

According to a preferred aspect of the invention, the HAS which is selectively oxidized at its reducing end, and thus one or more carboxylic acids in the oxidized reducing end diester _{R B -O- (C = O)} -OR C ( where, R _B And R < _C > may be the same or different. Preferably, in this way, a reactive HAS according to the following formula is obtained:

In the above, HAS 'is preferably HES'.

Suitable carbonic acid diester compounds are those wherein the alcohol component thereof is independently selected from the group consisting of N-hydroxysuccinimides such as N-hydroxysuccinimide or sulfo-N-hydroxysuccinimide, For example, p-nitrophenol, o, p-dinitrophenol, o, o'-dinitrophenol, trichlorophenol such as 2,4,6-trichlorophenol or 2,4,5-trichloro Phenol, trifluorophenol such as 2,4,6-trifluorophenol or 2,4,5-trifluorophenol, pentachlorophenol, pentafluorophenol, or hydroxyazole, such as, for example, A compound that is a compound that is Roxy benzotriazole can be used. N, N'-disuccinimidyl carbonate and sulfo-N, N'-disuccinimidyl carbonate are particularly preferred, and N, N'-disuccinimidyl carbonate is particularly preferred.

Thus, the present invention also relates to a method as described above, wherein the HAS selectively oxidized at its reducing end is activated by reacting the oxidized HAS with N, N'-disuccinimidyl carbonate.

The acidic alcohol is reacted with the salt of oxidized HAS or oxidized HAS at a molar ratio of acidic alcohol: HAS of preferably from 5: 1 to 50: 1, more preferably from 8: 1 to 20: 1, Deg.] C, preferably 10 to 30 [deg.] C, particularly preferably 15 to 25 [deg.] C. The reaction time is preferably 1 to 10 h, more preferably 2 to 5 h, more preferably 2 to 4 h, particularly 2 to 3 h.

The carbonic acid diester compound is reacted with a salt of oxidized HAS or oxidized HAS, generally in a molar ratio of diester compound: HAS of 1: 1 to 3: 1, for example 1: 1 to 1.5: 1. The reaction time is generally from 0.1 to 12 h, for example from 0.2 to 6 h, or from 0.5 to 2 h or from 0.75 to 1.25 h.

According to a preferred embodiment of the present invention, the reaction of the oxidized HAS with an acidic alcohol and / or a carboxylic acid diester is carried out in the presence of at least one aprotic solvent, for example a water content of 0.5% by weight or less, preferably 0.1% In an anhydrous aprotic solvent. Suitable solvents are, in particular, dimethylsulfoxide (DMSO), N-methylpyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF) and mixtures of two or more thereof. The reaction temperature is preferably in the range of 2 to 40 占 폚, more preferably in the range of 10 to 30 占 폚.

In order to react the oxidized HAS with one or more acidic alcohols, one or more additional activators are used.

Suitable activating agents are, in particular, carbonyldiimidazole, carbodiimides such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-ethyl-3- ) Carbodiimide (EDC), with dicyclohexylcarbodiimide (DCC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) being particularly preferred.

The invention therefore also relates to a process as mentioned above, wherein the HAS oxidized at its reducing end is reacted with an acidic alcohol in the presence of an additional activator to obtain a reactive HAS ester.

According to one aspect of the present invention, the reaction of the oxidized HAS with a carbonic acid diester and / or an acidic alcohol can be carried out in the presence of a reducing agent, Is carried out under basic activity. Prior to the addition, the water having little buffering agent had a pH value of 7 at 25 캜. After adding the reaction mixture and measuring the pH value, the base activity of the reaction mixture is obtained, which is preferably not more than 9.0, more preferably not more than 8.0, particularly preferably not more than 7.5.

According to another embodiment of the present invention, the oxidized HAS is reacted with EDC in the absence of water to react with N-hydroxysuccinimide in dry DMA to selectively obtain polymer N-hydroxysuccinimide ester according to the following formula :

More preferably, HAS 'is HES.

Such a reaction surprisingly suppresses the rearrangement reaction of O-acylisourea and oxidized HAS formed by EDC with respective N-acylurea without any byproducts resulting from the reaction of EDC with OH groups of HES do.

According to another preferred embodiment of the present invention, the oxidized HAS is reacted with N, N'-disuccinimidyl carbonate in anhydrous DMF in the absence of water and in the absence of an activator to form HAS-N- Lt; RTI ID = 0.0 > of:

More preferably, HAS 'is HES.

According to another embodiment of the present invention, the HAS selectively oxidized at its reducing end is reacted with an azole at the oxidized reducing end, for example, with carbonyldiimidazole or carbonyl dibenzimidazole to give a polymer having a reactive carboxy group . In the case of carbonyldiimidazole, a reactive imidazolide HAS derivative according to the following formula is produced:

In the above, HAS 'is preferably HES'.

The reactive HAS derivative comprising at least one reactive carboxy group, which is preferably produced by reacting HAS with an acidic alcohol, carbonate and / or azolide as mentioned above, is further reacted with the amino group M of the cross-linking compound .

Reaction of the HAS with the amino group M through an optionally further activated oxidized reducing end as mentioned above can be carried out according to any suitable method. Preferably, the amino group M is a primary amino group H ₂ N- or a secondary amino group.

Generally, it is preferred to use a polar aprotic solvent which may contain a certain amount of water, for example less than or equal to 10% by weight of water. Preferred aprotic solvents are, in particular, DMSO or DMF.

A specific example of the preferable reaction temperature range is 0 to 80 캜, more preferably 0 to 70 캜, more preferably 0 to 60 캜, more preferably 0 to 50 캜, even more preferably 0 to 40 캜.

When a crosslinking compound is used to react with a HAS having an oxidized form of a reducing end having H ₂ N- as the amino group M according to a preferred embodiment, the HAS derivative is crosslinked with the HAS used as the starting material (I) of the present invention in which the compound is linked via an amide bond, the HAS derivative thus obtained further contains an acetal or keto group A.

Thus, the present invention also relates to a process for the preparation of a compound of formula (I) wherein (i) HAS is reacted with an amino group M of the crosslinking compound via an oxidized reducing end thereof, wherein M is H ₂ N-, And X is - (C = O) -NH-. ≪ / RTI >

Accordingly, the present invention also relates to a HAS derivative obtainable or obtained by the above-mentioned method.

Further, the present invention relates to a HAS derivative per se having the following structure:

In the above, R 'is H when the amino group M of the crosslinkable compound is a primary amino group, and R' is a chemical moiety other than H when the amino group M of the crosslinkable compound is a secondary amino group. Since the exact chemical nature of R 'depends on the crosslinking compound, reference can be made to the discussion of the crosslinking compound which is generally possible and preferred.

According to the above-mentioned preferable crosslinking compounds mentioned in the present invention, the following HAS derivatives may be mentioned as preferred embodiments, for example, in each case HAS is HES according to a preferred embodiment of the present invention:

or

or

또는

or

또는

또는

또는

또는

또는

또는

또는

또는

또는

또는

또는

또는

또는

or

or

or

or

or

or

or

or

or

or

or

or

or

or

A HAS derivative based on a crosslinking compound selected from the group consisting of structures (a2), (a4), (a11), (a12), (a14), (a16), and (a18) is more preferable. More preferred are HAS derivatives based on a crosslinking compound selected from the group consisting of structures (a2), (a11), (a12), (a14), (a16), and (a18). Particularly preferred are HAS derivatives based on crosslinking compounds selected from the group consisting of structures (a2), (a11), (a12) and (a16).

According to a particularly preferred embodiment, the present invention relates to a HES derivative having the structure:

Even more preferably, the HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, more preferably about 2 to about 400 kDa, More preferably from about 10 to about 200 kDa, especially from about 50 to about 150 kDa, with a molar substitution of from 0.1 to 3, preferably from 0.4 to 1.3, such as 0.4, 0.5, 0.6, 0.7, The ratio of C ₂ : C ₆ substitution is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, even more preferably in the range of 3 to 12.

The non-oxidized reducing end

According to a second and preferred embodiment of the present invention, a crosslinking compound is reacted with an HAS, preferably a non-oxidized C * atom of a reducing end of HES, via an amino group, i.e. the terminal aldehyde group of the HAS molecule is reacted with an aldehyde group And may exist in the form of a corresponding and / or corresponding hemiacetal.

The reaction of the HAS with the amino group M through the non-oxidized reducing end can be carried out according to any suitable method. Preferably, the amino group M is H ₂ N-, a suitable secondary amino group HNR'-, for example H ₃ C-NH-, H ₂ NO-, or a suitable secondary hydroxyamino group HNR'-O-, For example, H ₃ C-NH-O- or H ₂ N-NH- (C = O) -.

Preferably, the amino group M is H ₂ N-, H ₂ NO- or H ₂ N-NH- (C═O) -, more preferably H ₂ N- or H ₂ NO-, especially H ₂ N- .

According to a preferred embodiment of the invention, the reaction is carried out in an aqueous system. The term "aqueous system " as used in the context of the present invention refers to an aqueous system comprising at least 10% by weight, preferably at least 50% by weight, more preferably at least 80% by weight, based on the weight of the solvent, More preferably 90% by weight or more, or 100% by weight or less. As an additional solvent, a solvent such as DMSO, DMF, ethanol or methanol may be mentioned.

According to a preferred embodiment, when the HAS is reacted with a crosslinking compound in an aqueous medium and the amino group M of the crosslinking compound is hydroxylamine or hydrazide, the reaction temperature is preferably in the range of 5 to 45 ° C, Is in the range of 10 to 30 占 폚, particularly preferably in the range of 15 to 25 占 폚.

According to another preferred embodiment, when the HAS is reacted with a crosslinking compound in an aqueous medium and the amino group M of the crosslinking compound is a group H ₂ N- or R'HN-, the reaction is a reductive amination reaction, The temperature is preferably in the range of 100 占 폚 or less, more preferably in the range of 10 to 90 占 폚, more preferably in the range of 20 to 80 占 폚, more preferably in the range of 30 to 70 占 폚, particularly preferably in the range of 40 to 60 占 폚.

During the course of the reaction, the temperature may preferably vary within the ranges given above, or may be kept substantially constant.

The reaction time required to react the HAS with the group M of the crosslinking compound can be adapted to meet the specific needs, generally ranging from 1 h to 7 d. For example, when the amino group M is hydroxylamine or hydrazide, the reaction time is preferably in the range of 1 h to 3 d, more preferably 2 h to 48 h, particularly preferably 3 to 24 h.

For example, when the reaction of the HAS and the crosslinkable compound is a reductive amination reaction, the reaction time is preferably in the range of 1 h to 7 d, more preferably in the range of 4 h to 6 d, more preferably 8 h To 5 d, even more preferably from 16 h to 3 d.

The pH value for the reaction of the HAS with the crosslinkable compound can be adapted to meet the specific needs, such as the chemical nature of the reactants. For example, when the group M of the crosslinkable compound is hydroxylamine or hydrazide, the pH value is preferably in the range of 3 to 9, more preferably 4 to 8, even more preferably 4.5 to 6.5.

For example, when the reaction of the HAS and the crosslinkable compound is a reductive amination reaction, the pH value is preferably in the range of 3 to 9, more preferably 3.5 to 8, even more preferably 4 to 6.

Suitable pH values of the reaction mixture can be adjusted for each reaction step by adding one or more suitable buffers. As a preferred buffer, mention may be made of acetate buffers, preferably sodium acetate buffers, phosphates or borate buffers.

When the crosslinking compound is used in the reaction with HAS having a non-oxidized form of reducing end having H ₂ N- as an amino group M according to a preferred embodiment, the HAS derivative is crosslinked with the HAS used as a starting material (I) of the present invention in which the compound is bonded via an imine bond, the HAS derivative thus obtained further contains an acetal or keto group A. When this reaction is carried out under reductive amination conditions in the presence of a suitable reducing agent, the HAS derivative is obtained by step (i) of the present invention which links the crosslinkable compound with the HAS used as the starting material through an amine linkage , The HAS derivative thus obtained further contains an acetal or keto group A.

Thus, the invention also relates to a process for the preparation of a compound of formula (I) wherein, in step (i), the HAS is reacted with the amino group M of the crosslinking compound, wherein M is H ₂ N-, via its non- And wherein the reaction is carried out at a temperature ranging from 20 to 80 캜 at a pH in the range of from 4 to 7, wherein X is -CH = N-.

In addition, the present invention also relates to a process for the preparation of compounds of formula (I) wherein, in step (i), the reaction is carried out in the presence of a reducing agent such as sodium borohydride, sodium cyanoborohydride, organoborane complex compounds such as 4- (dimethylamine) pyridine borane complex, N-ethyldiisopropylamine borane complex, N-ethylmorpholine borane complex, N-methylmorpholine borane complex, N-phenylmorpholine borane complex, lutidine borane complex, triethylamine borane complex, or trimethylamine borane complex , Preferably in the presence of NaCNBH ₃ , to obtain a HAS derivative, wherein X is -CH ₂ -NH-.

The concentration of these reducing agents used in the reductive amination reaction of the present invention is preferably in the range of 0.01 to 2.0 mol / l, more preferably 0.05 to 1.5 mol / l, based on the volume of the reaction solution in each case , More preferably in the range of 0.1 to 1.0 mol / l.

According to the above-mentioned preferred embodiment wherein M is H ₂ N- and the reaction of the crosslinkable compound with HAS is carried out under reductive amination conditions, the molar ratio of the crosslinking compound: HAS is preferably from 1: 1 to 100: 1 , More preferably from 2: 1 to 80: 1, more preferably from 3: 1 to 70: 1, even more preferably from 4: 1 to 60: 1, and still more preferably from 5: 1 to 50:

According to the above-mentioned preferred embodiment wherein M is H ₂ N- and the reaction of the cross-linking compound with HAS is carried out under reductive amination conditions, the concentration of HAS, preferably HES, in the aqueous system, %, More preferably 3 to 45 wt .-%, more preferably 5 to 40 wt .-%, based on the weight of the composition.

When a crosslinking compound is used in the reaction with HAS having a non-oxidized form of a reducing end having H ₂ NO- or H ₂ N-NH- (C═O) - as an amino group M according to a preferred embodiment , The HAS derivative is prepared by step (i) of the present invention in which the HAS used as the starting material is linked to the crosslinkable compound via a -CH = NO- or -CH = N-NH- (C = O) , Wherein the resulting HAS derivative further contains an acetal or keto group A. When this reaction is carried out under reducing conditions in the presence of a suitable reducing agent, the HAS derivative is reacted with the HAS used as the starting material by reacting the crosslinking compound with a -CH ₂ -NH-O- linkage or -CH ₂ -NH-NH- ( C = O) - bond, wherein the HAS derivative thus obtained further contains an acetal or keto group A.

Thus, the present invention also relates to a process for the preparation of a compound of formula (I) wherein, in step (i), the HAS is reacted with an amino group M of a crosslinking compound, wherein M is H ₂ NO- or H ₂ N -NH- (C = O) - and the reaction is carried out at a temperature in the range of from 5 to 80 C at a pH in the range of from 4.5 to 6.5, where X is -CH = NO- or -CH = N-NH- (C = O) -), as described above.

In addition, the present invention also relates to a process for the preparation of compounds of formula (I) wherein, in step (i), the reaction is carried out in the presence of a reducing agent such as sodium borohydride, sodium cyanoborohydride, organoborane complex compounds such as 4- (dimethylamine) pyridine borane complex, N-ethyldiisopropylamine borane complex, N-ethylmorpholine borane complex, N-methylmorpholine borane complex, N-phenylmorpholine borane complex, lutidine borane complex, triethylamine borane complex, or trimethylamine borane complex , Preferably in the presence of NaCNBH ₃ , to give a HAS derivative, wherein X is -CH ₂ -NH-O- or -CH ₂ -NH-NH- (C = O) -, It is about the same method.

The concentration of these reducing agents used in the reaction of the present invention is preferably in the range of 0.001 to 2.0 mol / l, more preferably in the range of 0.01 to 1.0 mol / l, in each case based on the volume of the reaction solution, And more preferably in the range of 0.1 to 0.8 mol / l.

According to the above-mentioned preferred embodiment wherein M is H ₂ NO- or H ₂ N-NH- (C = O) - and the reaction of the crosslinkable compound with HAS under reducing conditions, the crosslinking compound: HAS The molar ratio is preferably from 1: 1 to 100: 1, more preferably from 2: 1 to 80: 1, more preferably from 3: 1 to 70: 1, even more preferably from 4: 1 to 60: 1 to 50: 1.

According to the above-mentioned preferred embodiment wherein M is H ₂ N- and the reaction of the cross-linking compound with HAS is carried out under reductive amination conditions, the concentration of HAS, preferably HES, in the aqueous system, %, More preferably 3 to 45 wt .-%, more preferably 5 to 40 wt .-%, based on the weight of the composition.

Accordingly, the present invention also relates to HAS derivatives obtainable or obtained by the method (s) as mentioned above.

Further, the present invention also relates to

(Wherein the C-N double bond may be present in E or Z stereochemistry, depending on the reaction conditions and / or the specific chemical nature of the crosslinking compound, there may be a mixture of both forms with a particular equilibrium distribution); or

For the purposes of the present invention, so long as the corresponding ring structure to be considered as being equal to the open structure is concerned,

Depending on the reaction conditions and / or the specific chemical nature of the crosslinking compound, the HAS derivative may be present in the equatorial or axial position with the N-atom, and there may be a mixture of both forms with a particular equilibrium distribution have); Or structure

or

Or structure

또는

or

or

Or a corresponding cyclic structure

or

Lt; RTI ID = 0.0 > HAS < / RTI >

According to the preferred crosslinking compounds mentioned above, the following HAS derivatives may be mentioned as preferred embodiments, in each case HAS being a HES according to a preferred embodiment of the invention:

(Wherein the corresponding ring structure is included); or

or

(Wherein the corresponding ring structure is included); or

or

(여기서, 상응하는 환 구조가 포함된다); 또는

(Wherein the corresponding ring structure is included); or

(여기서, 상응하는 환 구조가 포함된다); 또는or

(Wherein the corresponding ring structure is included); or

or

(Wherein the corresponding ring structure is included); or

or

or

(Wherein the corresponding ring structure is included); or

(Wherein the corresponding ring structure is included); or

or

(Wherein the corresponding ring structure is included); or

or

(Wherein the corresponding ring structure is included); or

or

(Wherein the corresponding ring structure is included); or

(여기서, 상응하는 환 구조가 포함된다); 또는or

(Wherein the corresponding ring structure is included); or

또는

(여기서, 상응하는 환 구조가 포함된다); 또는

or

(Wherein the corresponding ring structure is included); or

(여기서, 상응하는 환 구조가 포함된다); 또는or

(Wherein the corresponding ring structure is included); or

(여기서, 상응하는 환 구조가 포함된다); 또는or

(Wherein the corresponding ring structure is included); or

(여기서, 상응하는 환 구조가 포함된다); 또는or

(Wherein the corresponding ring structure is included); or

(여기서, 상응하는 환 구조가 포함된다); 또는or

(Wherein the corresponding ring structure is included); or

A HAS derivative based on a crosslinking compound selected from the group consisting of structures (a2), (a4), (a11), (a12), (a14), (a16), and (a18) is more preferable. More preferred are HAS derivatives based on a crosslinking compound selected from the group consisting of structures (a2), (a11), (a12), (a14), (a16), and (a18). Particularly preferred are HAS derivatives based on crosslinking compounds selected from the group consisting of structures (a2), (a11), (a12) and (a16).

According to a particularly preferred embodiment,

The corresponding ring structure

And / or

≪ / RTI > More preferably, the HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, more preferably about 2 to about 400 kDa, More preferably about 10 to about 200 kDa, especially about 50 to about 150 kDa, and the molar substitution is from 0.1 to 3, preferably 0.4 to 1.3, such as 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3, and the C ₂ : C ₆ substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15,

Other modes of thinking about HAS

To be perfect, it should be noted that although not preferred according to the present invention, it may be envisaged to oxidize HAS before it is reacted with the crosslinking compound so that two or more aldehyde groups are introduced into the HAS according to the following formula :

Generally, a combination of each oxidizing agent or oxidizing agent capable of oxidizing one or more saccharide rings of the polymer to provide an open saccharide ring having one or more, preferably two or more, aldehyde groups may be used. This reaction can be illustrated by the following reaction scheme showing the saccharide ring of HAS oxidized to provide an open ring with two aldehyde groups:

Suitable oxidizing agents are, in particular, periodic iodides, for example alkali metal and iodides or mixtures of two or more thereof, with sodium periodate and potassium periodate being preferred. It can be considered that these aldehyde groups can be reacted with the crosslinkable compound M-L-A via the amino group M.

Isolation and / or purification

In general, the HAS derivative from step (i) of the present invention may be considered to be subjected to a subsequent reaction as described below. According to a preferred embodiment, it is suitable to purify the HAS derivative from step (i) after reaction step (i).

To purify the HAS derivative from step (i), for example, the following possibilities A) to C) may be mentioned, possibility A) being preferred:

A) concentration is preferably 0.1 to 100 mmol / l, more preferably 1 to 50 mmol / l, more preferably 5 to 20 mmol / l, such as about 10 mmol / l, , More preferably 4 to 10, more preferably 6 to 10, and even more preferably 8 to 10, for example, about 9, or an ultrafiltration method using water (the number of exchange cycles is preferably 10 to 50 , More preferably 10 to 40, even more preferably 10 to 30, such as 20).

B) concentration is preferably 0.1 to 100 mmol / l, more preferably 1 to 50 mmol / l, more preferably 5 to 20 mmol / l, such as about 10 mmol / l, , More preferably from 4 to 10, more preferably from 6 to 10, and even more preferably from 7 to 10, or water, wherein the HAS derivative is contained at a desired concentration of 2 to 20 wt% Solution, and the buffer or water is used in a particularly excessive amount (about 100: 1) for the HES derivative solution.

C) precipitate using ethanol or isopropanol, centrifuge and redissolve in water to obtain a solution having a preferred concentration of about 10% by weight; The concentration is preferably 0.1 to 100 mmol / l, more preferably 1 to 50 mmol / l, more preferably 5 to 20 mmol / l, such as about 10 mmol / l and the pH is preferably 2 to 10 (The number of exchange cycles is preferably from 10 to 40, more preferably from 10 to 40, more preferably from 10 to 40, more preferably from 10 to 40, most preferably from 10 to 40, 30, still more preferably 10 to 20, for example 10).

After the preferred purification step, the HAS derivative is preferably obtained as a solid. According to a further conceivable embodiment of the invention, the preferred HAS derivative content is from 2 to 40% by weight, the pH of the solution is preferably in the range from 3 to 10 and the concentration of the buffer used is preferably from 0.1 to 1 mol / l Of the HAS derivative solution or the solution of the freeze HAS derivative.

Therefore, the present invention also relates to a method for preparing a HAS derivative obtained in (i) after (i) by an ultrafiltration method using an aqueous buffer solution or water having a concentration of 0.1 to 100 mmol / l and a pH in the range of 2 to 10 (The number of exchange cycles is 10 to 50). ≪ / RTI >

Reaction with biologically active agent BA

According to a further preferred embodiment, the present invention relates to the above-mentioned HES derivative

Is further suitably reacted with the biologically active compound BA through an acetal or ketal group A, which is preferably converted to the corresponding aldehyde or keto group before the reaction with BA.

Most preferably, the group A, preferably the corresponding aldehyde or keto group, is reacted with an amino group, more preferably a primary amino group contained in BA. In this case and for the purposes of the present invention, BA is also represented as H ₂ N-BA ', wherein BA' is the remainder of BA.

Therefore, the present invention also relates to

(ii) reducing amination of the HAS derivative according to formula III with an amino group of the biologically active agent H ₂ N-BA 'through group A to give a HAS derivative according to formula IV

, ≪ / RTI > further comprising the steps of:

(IV)

According to the first aspect of the present invention, preferably, the HAS derivative obtained from purified (i) is suitably converted into a corresponding aldehyde or keto group by reacting the resulting HAS derivative with BA And is suitably purified and / or isolated prior to administration. The conversion to an aldehyde or keto group is preferably carried out by an acid-catalyzed hydrolysis reaction. This reaction is preferably carried out at a temperature of from 0 to 100 캜, more preferably from 10 to 80 캜, more preferably from 20 to 60 캜; Preferably at a pH in the range of 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 3. Purification and buffer-exchange of hydrolysis reaction products can be accomplished by methods well known to those skilled in the art, e. G., Dialysis or ultrafiltration. The converted material can be recovered as a solid from solution by, for example, lyophilization.

According to the second aspect of the present invention, preferably, the HAS derivative obtained from purified (i) is suitably converted into a corresponding aldehyde or keto group by converting the group A into a corresponding HAS derivative, , I.e. no separate purification and / or isolation steps are carried out on HAS derivatives containing aldehydes or keto groups. The conversion to an aldehyde or keto group is preferably carried out by an acid-catalyzed hydrolysis reaction. This reaction is preferably carried out at a temperature of from 0 to 100 캜, more preferably from 10 to 80 캜, more preferably from 20 to 60 캜; Preferably at a pH in the range of 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 3. The hydrolysis reaction product can be combined with BA either directly or after adjusting the pH to a value that is compatible with the reaction with the BA.

Thus, the present invention also relates to (ii) a process as described above, wherein the group A of the HAS derivative according to formula (III) is previously converted to the corresponding aldehyde or keto group.

According to a third aspect of the present invention, the HAS derivative obtained preferably from purified (i) is reacted directly with BA, i.e. without carrying out a separate suitable purification and / or isolation step and by reacting the group A with the corresponding aldehyde Or reacted with BA under reaction conditions which allow the conversion of group A into the corresponding aldehyde or keto group without extra steps to convert it to a keto group. The conversion to an aldehyde or keto group is preferably carried out by an acid-catalyzed hydrolysis reaction. This reaction is preferably carried out at a temperature of from 0 to 100 캜, more preferably from 10 to 80 캜, more preferably from 20 to 60 캜; Preferably at a pH in the range of 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 3. The hydrolysis reaction product can be combined with BA either directly or after adjusting the pH to a value that is compatible with the reaction with the BA.

The manner in which the method according to the third aspect mentioned above is carried out depends, for example, on the specific properties of the biologically active substance BA used. For example, when a protein such as EPO, G-CSF or IFN alpha is used as the BA, the above-identified first or second aspect is generally suitable.

The reaction in step (ii) is preferably carried out in an aqueous system. The term "aqueous system " as used in this context of the present invention refers to an aqueous system comprising at least 10% by weight, preferably at least 50% by weight, more preferably at least 80% by weight, based on the weight of the solvent, By weight or more, still more preferably 90% by weight or more, or 100% by weight or less. As an additional solvent, a solvent such as DMSO, DMF, ethanol or methanol may be mentioned.

Although it is conceivable to carry out the reaction in step (ii) under the conditions to obtain the non-reduced form of the HAS derivative according to formula (IV), the reaction according to step (ii) is carried out in the presence of one or more suitable reducing agents It is particularly preferred to carry out under reductive amination conditions:

Formula IV

In particular, under these conditions, the group -CH = N- obtained by reacting an aldehyde or keto group generated from group A of the HAS derivative with a H ₂ N group of BA is reduced to give -CH ₂ -NH-.

For example, the following reducing agents can be used: NaBH (OAc) ₃ , sodium borohydride, sodium cyanoborohydride, organoborane complex compounds such as 4- (dimethylamine) pyridine borane complex, N- N-ethylmorpholine borane complex, N-methylmorpholine borane complex, N-phenylmorpholine borane complex, lutidine borane complex, triethylamine borane complex, or trimethylamine borane complex. NaBH ₄ and NaCNBH ₃ are preferred, and NaCNBH ₃ is particularly preferred.

In one embodiment, the HAS derivative is added to an aqueous solution containing the biologically active agent. Preferably, a succession of one or more reducing agents, in particular NaCNBH _3.

In an alternative embodiment, the HAS derivative may optionally be introduced into an aqueous solution, followed by addition of BA. Preferably, a succession of one or more reducing agents, in particular NaCNBH _3.

The reaction of the HAS derivative with the amino group of the biologically active compound BA in step (ii) is preferably carried out at a pH of from 3 to 9, preferably from 3 to 8, more preferably from 3 to 7, even more preferably from 3 to 7, Lt; / RTI > is carried out at a pH of 3 or 4 or 5 or 6. Suitable pH values of the reaction mixture can be adjusted by adding one or more suitable buffers. As a preferred buffer, mention may be made of acetate buffers, preferably sodium acetate buffers, phosphates or borate buffers.

The reaction of the HAS derivative obtained in step b) with the amino group of the biologically active compound BA in step (ii) is preferably carried out at a temperature of -10 to 100 DEG C, preferably 0 to 50 DEG C, more preferably 0 to 37 DEG C, For example 0, 5, 10, 15, 20, or 25 < 0 > C.

The reaction time in step (ii) depends on the temperature, the HAS, in particular the ratio of the HES derivative to the compound BA, and the absolute concentration of the compound HAS and the compound BA. In general, a reaction time of from 5 minutes to 7 days, preferably from 1 hour to 7 days, is conceivable.

The molar ratio of the HAS derivative obtained in step (ii) to the compound BA is preferably 0: 1 to 200: 1 equivalent based on the number average molecular weight (M _n ) of the HAS derivative, more preferably 1: : 1. Preferably, the molar ratio is 1: 1 to 50: 1. For example, in the case where BA is a protein, particularly IFN alpha, a low molar ratio, for example, 50: 1 or less, preferably 1: 1 to 20: 1, more preferably 1: 1 to 10: To 2: 1 to 9: 1, more preferably from 3: 1 to 8: 1, even more preferably from 4: 1 to 7: 1.

In a particularly preferred embodiment, the concentration of the HAS derivative used in step (ii) is greater than about 10% by weight, in particular greater than about 15% by weight, based on the weight of the reaction solution of (ii) in each case.

Thus, the invention also relates to a process for the preparation of a compound of formula (I) wherein, in step (ii), the reaction is carried out in the presence of a reducing agent, preferably NaCNBH ₃ , preferably in an aqueous system, at a temperature in the range from 0 to 37 캜, preferably in the range from 0 to 25 캜, (Ii), the molar ratio of the HAS derivative to the biologically active agent BA is from 0.1: 1 to 200: 1 equivalent based on the number average molecular weight (M _n ) of the HAS derivative, and 1: 1 to 50: 1 < RTI ID = 0.0 > equivalents. ≪ / RTI >

A preferred concentration of BA, for example a solution, preferably an aqueous solution, of (ii) is in the range of 0.1 to 10 g / l, more preferably 1 to 9 g / l. (w / v)] of the HAS derivative in the solution before the step (ii) is preferably in the range of 0.1 to 50%, more preferably 0.5 to 45%, and still more preferably 1 to 40%.

According to a conceivable embodiment, the biologically active agent BA can be dissolved in an aqueous medium, preferably an aqueous buffer solution, in particular a sodium acetate buffer solution. Such an aqueous solution may additionally comprise additives such as, for example, SDS, Chaps, Tween 20, Tween 80, Nonidet P-40, and Triton X 100 ≪ / RTI > and / or < / RTI > When a disintegrant and / or dispersant is used, it is preferably present in an amount of 0.005 to 3% by weight, preferably 0.05 to 3% by weight, preferably about 0.5% by weight, based on the total weight of the aqueous solution.

One or more reducing agents are used in accordance with the invention and X present in the HAS derivative used in the reaction with BA is, for example, -CH = N-, -CH = NO-, or -CH = N-NH- = O) -, it is preferably reduced under reductive amination conditions used in the reaction of (ii) to give -CH ₂ -NH-, -CH ₂ -NH-O-, or -CH ₂ - NH-NH- (C = O) -.

Accordingly, the invention relates to a process as mentioned above, in particular in (ii), the reaction is carried out in the presence of a reducing agent, preferably NaCNBH ₃ , preferably in an aqueous system, at a temperature in the range from 0 to 37 ° C, preferably in the range from 0 to 25 ° C And at a pH in the range of from 3 to 9, more preferably from 3 to 7 and more preferably from 3 to 7, and in (ii) the molar ratio of the HAS derivative to the biologically active agent BA is lower than that of the HAS derivative used in step (ii) to be based on an average molecular weight (M _n) and from 0.1: 1 to 200: 1 equivalents, preferably from 1: 1 to 10: 1 equivalents (in each case, with the proviso that X is not a group is an amide), the above-mentioned as To a HAS derivative obtainable or obtainable by the method. A particularly preferred HAS derivative to biologically active agent BA molar ratio is less than 10, such as 1: 1 to 9: 1, more preferably 1: 1 to 8: 1, more preferably 1: 1 to 7: 1: about 1: 1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1. Although such low molar ratios, for example molar ratios of up to 50: 1, more preferably up to 10: 1, are conceivable for some biologically active agents, it is possible, for example, . For example, as long as G-CSF and EPO are involved, a molar ratio of 50: 1 or less, for example, 1: 1 to 50: 1 is preferred. In general, mention may be made of a molar ratio of, for example, 1: 1 to 50: 1 or 2: 1 to 40: 1 or 3: 1 to 30: 1 or 4: 1 to 20: 1 or 5: can do.

When the protein is used as the biologically active agent BA according to the invention and is particularly used at the abovementioned preferred pH range, in particular at a pH of less than or equal to 3, the HAS derivative mainly reacts with the amino group located at the N-terminus of the protein. The term "predominant" as used in the context of the present invention means that at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85% Lt; RTI ID = 0.0 > reductive amination. ≪ / RTI > More than 90%, or at least 95%, or at least 96% or at least 97% or at least 98% or at least 99% of the available N-terminal amino groups can be reacted. Although coupling with an amino group other than the N-terminal amino group could not be completely dispensed, coupling through reductive amination according to the present invention at a pH of less than 7 is believed to occur almost exclusively at the N-terminal amino group . In particular, these reaction conditions are preferable for stable proteins under these conditions. If the protein is, for example, acid labile, for example alpha-1-antitrypsin (A1AT), it is preferred to select the appropriate reaction conditions, in particular a pH of less than 7.5 to more than 5.

Thus, the present invention also relates to a process for the preparation of a HAS derivative comprising BA ', a HAS derivative obtainable or obtained by said process, and a HAS derivative according to formula IV as mentioned above, A HAS derivative comprising an N-terminal amino group and at least one additional amino group, wherein the HAS is coupled with an N-terminal amino group.

Accordingly, the present invention also relates to a HAS derivative according to formula IV:

Formula IV

Furthermore, the present invention also relates to hydroxyalkyl starch derivatives of formula (IV)

Formula IV

Wherein X is a carbon atom C * of a hydroxyalkyl starch (HAS) of formula I, wherein C * is optionally oxidized, most preferably HAS and M (Wherein X is not an amide group -C (= O) -NH-) via reaction with HAS of formula I through

Formula I

(II)

M-L-A

Wherein HAS 'is the remainder of the hydroxyalkyl starch molecule; R ₁ , R ₂ and R ₃ are independently hydrogen or a linear or branched hydroxyalkyl group;

A is a residue according to formula < RTI ID = 0.0 > Ha:

≪ RTI ID =

[In the above formula,

Z ₁ and Z ₂ are each independently O or S or NR _x , preferably O and R _x is H or lower alkyl such as methyl, ethyl or propyl, such as n- or i-propyl, or C (O) -R _y (where, R _y is preferably, C ₁ -C ₆ alkyl and C ₆ -C be selected from the group consisting of aryl and _14, a still more preferably, optionally substituted, preferably substituted Is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl; R _x is preferably H;

A ₁ and A ₂ are each independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, benzyl, 1,1,1- trichloroethyl, nitrobenzyl, methoxybenzyl Lt; / RTI > benzyl, or < RTI ID = 0.0 >

≪ RTI ID =

(From here,

A ₁ and A ₂ together are - (CH ₂ ) ₂ - or - (CH ₂ ) ₃ - or - (CH ₂ CH (CH ₃ )

A ₃ is H or methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, benzyl or together with the N atom of the amino group M or together with the appropriate atom , A < ₃ > is preferably H);

L is a spacer bridging M and A};

BA 'is the remainder of the biologically active agent BA'-NH ₂ remaining after reacting the amino group of BA with A via reductive amination or reacting with the corresponding aldehyde or ketone group of A.

For preferred embodiments relating to HAS, preferably HES, and crosslinkable compounds, reference is made to each of the above descriptions.

As far as the HAS derivatives of formula IV are concerned, it is preferred that X is preferably -CH ₂ -NH-, -CH═N-, -CH ₂ -NH-O- and -CH═N═O-, more preferably -CH ₂ -NH- and -CH ₂ -NH-O-, and most preferably -CH ₂ -NH-.

Furthermore, as far as the HAS derivative of the formula (IV) is concerned, L bridging M and A is preferably a spacer comprising at least one structural unit according to the formula (IId), preferably a structural unit according to the formula (IId)

≪ RTI ID =

In this formula,

L ₁ and L ₂ are, independently of each other, H or an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, alkyl Aryl, substituted alkylaryl and moiety -OR ", wherein R" is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, Aryl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl; Is preferably H or an organic moiety selected from the group consisting of alkyl and substituted alkyl; More preferably H or alkyl; More preferably H;

n is an integer of 1 to 20, preferably 1 to 10, more preferably 1 to 6, more preferably 1 to 4,

The term "biologically active material" (BA) as used in the context of the present invention refers to any biological or biological material It is about the material that can come in. In particular, the term "biologically active material ", as used in the context of the present invention, is intended to encompass a wide variety of biological or chemical agents intended to diagnose, cure, ameliorate, treat or prevent a disease in a human being or animal or to enhance the physical or mental well- Lt; / RTI > Examples of the active substance include oligonucleotides, polynucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, But are not limited to, nucleic acids, cells, viruses, liposomes, microparticles, and micelles. Preferably, the biologically active material according to the invention contains natural amino groups.

Examples of proteins include erythropoietin (EPO) such as recombinant human EPO (rhEPO) or an EPO-like agonist, a colony stimulating factor (CSF) such as G-CSF such as recombinant human G- Such as IFN alpha and IFN beta, such as recombinant human IFN alpha or IFN beta (rhIFN alpha or < RTI ID = 0.0 > 2 or IL-3 (such as rhIL-2 or rhIL-3), such as IL-2 or IL-3 or IL-11, Factor VII, Factor VII, Factor II, Factor III, Factor IV, Factor V, Factor VI, Factor X, Factor XI, Factor XI, Factor VIII, XII, Factor XIII, a serine protease inhibitor such as alpha-1-antitrypsin (A1AT), activating protein C (APC), a plasminogen activator, Such as recombinant human AT III (rhAT III), myoglobin, albumin, e.g., serum of bovine, such as bovine serum albumin, (BGF), brain-derived growth factor (BDGF), nerve growth factor (NGF), fibroblast growth factor (FGF) (BGF), B-cell growth factor (BGF), brain derived neurotrophic factor (BDNF), somatotropic factor (CNTF), transforming growth factors such as TGF alpha or TGF beta, BMP Hormones such as human growth hormone (hGH), such as recombinant human growth hormone (rhGH), tumor necrosis factors such as TNF alpha or TNF beta, somatostatine, somatotropin, Somatomedine, hemoglobin, hormone or pro-hormone, For example, insulin, gonadotropin, melanocyte stimulating hormone (alpha-MSH), tryptorelin, hypothalamic hormones such as antidiuretic hormone (ADH and oxytocin as well as release hormone and release- ), Parathyroid hormone, thyroid hormone such as thyroxine, thyrotropin, tyloribene, calcitonin, glucagon, glucagon-like peptide (GLP-1, GLP-2 etc.), exendin, Leptin, such as recombinant human leptin (rh-leptin), Kemptide (Trp ⁴ -camptid), vasopressin, gastrin, secretin, integrin integrin, glycoprotein hormones such as LH, FSH, etc., melanocyte stimulating hormone, lipoprotein and apolipoprotein such as apo-B, apo-E, apo-L _a , immunoglobulins such as IgG, IgE, IgM, IgA, IgD and fragments thereof, such as human immunoglobulin G (HFab), murine immunoglobulin G (mIgG), hirudin, tissue pathway inhibitors, plant proteins such as lectin or ricin, An immunotoxin, an antigen E, an alpha-proteinase inhibitor, a ragweed allergen inducing antigen, a melanin, an oligorhizin protein, an RGD protein, or any one of these proteins; Prolactin or a mutant thereof, such as G129R, wherein the wild-type amino acid glycine at position 129 is replaced by arginine (the trade name of such mutants is "LactoVert") and functional derivatives of these proteins or receptors Or fragments thereof, but are not limited thereto.

The polypeptides are preferably selected from the group consisting of erythropoietin (EPO) such as recombinant human EPO (rhEPO), a colony stimulating factor (CSF) such as G-CSF such as recombinant human G-CSF (rhG- (IFN), such as IFN alpha, IFN beta, IFN gamma, such as recombinant human IFN alpha (rhIFN alpha) or recombinant human IFN beta (rhIFN beta), Factor VII such as recombinant human Factor VIIa (rhFVIIa ), Factor IX, such as recombinant human factor IX (rhFIX), growth hormone (GH) such as recombinant human growth hormone (rhGH), Fab fragments such as Fab fragments derived from human immunoglobulin G molecules hFab), immunoglobulin G, such as murine immunoglobulin G (mIgG), glucagon-like peptide-1 (GLP-1), asparaginases such as recombinant asparaginase (rasparaginase) For example, recombinant human leptin (rh-leptin), interleukin-2, interleukin-11, alpha-1- Antitrypsin, antibody or antibody fragment, and alternative protein scaffolds.

More preferably, the polypeptide is selected from the group consisting of erythropoietin (EPO) such as recombinant human EPO (rhEPO), a colony stimulating factor (CSF) such as G-CSF such as recombinant human G-CSF (rhG- (IFN), such as IFN alpha, IFN beta, IFN gamma, such as recombinant human IFN alpha (rhIFN alpha) or recombinant human IFN beta (rhIFN beta), Factor VII, such as recombinant human Factor VIIa (e.g., rhFVIIa), Factor IX, such as recombinant human factor IX (rhFIX), growth hormone (GH), such as recombinant human growth hormone (rhGH), Fab fragments such as Fab fragments derived from human immunoglobulin G molecules (hFab), immunoglobulin G, such as murine immunoglobulin G (mIgG), glucagon-like peptide-1 (GLP-1), asparaginases such as recombinant asparaginase (rasparaginase), leptin , Such as recombinant human leptin (rh-leptin), interleukin-2, interleukin-11, Consisting of alpha-1-anti-trypsin is selected from the group.

The active substance is preferably selected from the group consisting of antibiotics, antidepressants, antidiabetic agents, antidiuretic agents, anticholinergics, antiarrhythmics, antagonists, antiinflammatory agents, antihistamines, antihistamines, antifungals, antimetabolites, antithrombotics, androgens, An estrogen, an antiestrogen, an anti-osteoporosis agent, an anti-tumor agent, a vasodilator, a therapeutic agent for hypertension, an antipyretic, an analgesic, an antiinflammatory agent, a beta blocker, an immunosuppressant and a vitamin.

Some additional non-limiting examples of active substances include alendronate, amikazin, atenolol, azathioprine, cimetidine, clonidine, kosinchlopin ( such as cycloserine, desmopressin, dihydroergotamine, dobutamine, dopamine, epsilon-aminocaproic acid, ergometrine, ss But are not limited to, esmolol, famotidine, flecainide, folic acid, flucytosine, furosemide, ganciclovir, glucagon, hydrazaline, iso But are not limited to, isoproterenol, ketamine, liothyronine, LHRH, merpatricin, methyldopa, metoprolol, neomicin, nimodipine, , Nystatin (nystatin), oxito The compounds of the present invention may be used in combination with other therapeutic agents such as oxytocin, phentolamine, phenylephrine, procainamide, procaine, propranolol, ritodrine, sotalol, Terbutaline, thiamine, tiludronate, tolazoline, trimethoprim, tromethamine, vasopressin; Aminoglutaric acid, aminocarboxylic acid, aminocarboxylic acid, aminosalicylic acid, aminocarboxylic acid, aminocarboxylic acid, aminocarboxylic acid, aminocarboxylic acid, aminocarboxylic acid, Anastrozole, asparaginase [e.g., recombinant asparaginase, e. Recombinant asparaginase (r asparaginase) from E. coli], anthracycline, bexarotene, bicalutamide, bleomycin, buserelin Carbusin, carmustine, chlorambucin, sodium cilastatin, sodium carboxymethylcellulose, carboxymethylcellulose, carboxymethylcellulose, carboxymethylcellulose, But are not limited to, cisplatin, cladribine, clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecin, 13-cis Retinoic acid, all trans retinoic acid; But are not limited to, dacarbazine, dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, , Doxorubicin, epirubicin, estramustine, etoposide, exemestane, fexofenadine, fludarabine, fludarocortisone (fludarabine, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine, L-dopa, hydroxyurea, idarubicin, , Ifosfamide, imatinib, irinotecan, itraconazole, goserelin, letrozole, leucovorin, levamisole, , Lisinopril, lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercuric acid, Methotrexate, metoclopramide, mexiletine, mitomycin, mitotane, mitoxanthrone, methotrexate, methotrexate, methotrexate, but are not limited to, mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, pilcamycin ), Porfimer, prednisone, procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus, streptavidin, The compounds of the present invention can be used in combination with streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, vinorelbine, dolasetron, granisetron; But are not limited to, formoterol, fluticasone, leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxin, A clooxide antiviral, aroyl hydrazone, sumatriptan; Macrolide, such as erythromycin, oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin, ), Azithromycin, flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin, leucomycin, myoin Miocamycin, rokitamycin, andazithromycin, and swinolide A; For example, fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norpuroxacin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin, pherophocassin, such as pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, Clinafloxacin, and sitafloxacin; For example, aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, And streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin, daptomycin, and gramicidin (see, for example, gramicidin, colistimethate; Polymixins such as polymyxin B, capreomycin, bacitracin, penem; Penicillins, such as penicillinase-sensitive agonists such as penicillin G, penicillin V; Penicillinase-resistant agents such as methicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; Gram negative microbial agonists such as ampicillin, amoxicillin, and hetacillin, cillin, and galampicillin; Antipseudomonal penicillins, such as carbenicillin, ticarcillin, azlocillin, mezlocillin, and piperacillin; Cephalosporins such as cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin, cephalosporin, ), Cephapirin, cephalexin, cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cephaloride, cefaclor, cefaclor, cefadroxil, cephaloglycin, cefuroxime, ceforamide, cefotaxime, cefatrizine, cephacetrile, Cefepime, cefixime, cefonicid, cell felazone, cefotetan, cefinetazole, ceftazidime, loracarbef, , And moxalactam, monobactam, such as aztreonam; And carbapenems such as imipenem, meropenem, pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenol (for example, metaproterenol sulphate, beclomethasone dipropionate, triamcinolone acetamide, budesonide acetonide, fluticasone, ipratropium bromide, fluney solid flunisolide, sodium cromolyn, and ergotamine tartrate; Taxanes such as paclitaxel; SN-38, and tyrphostine.

Thus, chemical compounds known to the person skilled in the art as "small molecules " are also biologically active substances which can be conceived according to the invention. The term "small molecule ", as used in the context of the present invention, relates to biologically active chemical compounds, including but not limited to proteins and oligonucleotides, containing peptides of up to 50 amino acids. A typical example of such a small molecule is listed in the preceding paragraph.

Examples for oligonucleotides are aptamers and siRNA. Also, peptide nucleic acid (PNA) is mentioned as a conceivable biologically active substance.

Thus, the present invention also relates to the use of recombinant human G-CSF (rhG), such as erythropoietin (EPO) such as recombinant human EPO (rhEPO), a colony stimulating factor (CSF) Such as recombinant human IFN alpha (rhIFN alpha) or recombinant human IFN beta (rhIFN beta), Factor VII, such as recombinant human (IFN) For example, human Factor VIIa (rhFVIIa), factor IX, such as recombinant human factor IX (rhFIX), growth hormone (GH), such as recombinant human growth hormone (rhGH), Fab fragments such as human immunoglobulin G molecules Like Fab fragments (hFab), immunoglobulin G, such as murine immunoglobulin G (mIgG), glucagon-like peptide-1 (GLP-1), asparaginases such as recombinant asparaginase ), Leptin such as recombinant human leptin (rh-leptin), interleukin-2, interleukin-11, Alpha-1-antitrypsin, an antibody or antibody fragment, and an alternative protein scaffold, as described above, and a HAS derivative as mentioned above.

The term "alternative protein scaffold " as used in the context of the present invention relates to a molecule having binding capacity similar to that of a given antibody, which molecule is based on an alternative non-antibody protein backbone. In this context, reference can be made to the literature (see, for example, A. Skerra, T. Hey et al., And H. K. Binz (see reference list).

Insofar as the biologically active substance (BA) of the present invention is concerned, these compounds may contain one or more amino groups for coupling according to step (ii) of the present invention. When BA itself does not include an amino group suitable for such coupling, one or more of these amino groups may be suitably functionalized by methods known to those skilled in the art, prior to performing (ii) on BA BA. ≪ / RTI >

According to the above mentioned biologically active agents, in particular the above-mentioned preferred biologically active agents, and according to the above-mentioned preferred cross-linking compounds and the HAS derivatives obtained therefrom, the following HAS derivatives may be mentioned, for example, as preferred embodiments, In each case the HAS is a HES according to a preferred embodiment of the invention:

또는

또는

또는

또는

or

or

or

or

또는

또는or

or

or

또는

또는

또는

또는

또는

또는

또는

or

or

or

or

or

or

or

                                                                         or

In the above, BA 'is a protein, and more preferably such a protein is selected from the group consisting of erythropoietin (EPO) such as recombinant human EPO (rhEPO), a colony stimulating factor (CSF) such as G- CSF, Such as human G-CSF (rhG-CSF), interferon (IFN) such as IFN alpha, IFN beta, IFN gamma such as recombinant human IFN alpha (rhIFN alpha) or recombinant human IFN beta (rhIFN beta) Such as recombinant human factor VIIa (rhFVIIa), Factor IX, such as recombinant human factor IX (rhFIX), growth hormone (GH), such as recombinant human growth hormone (rhGH) (HFab) derived from an immunoglobulin G molecule, immunoglobulin G, e.g., murine immunoglobulin G (mIgG), glucagon-like peptide-1 (GLP-1), asparaginases such as recombinant asparagine (R asparaginase), leptin, such as recombinant human leptin Tin), interleukin-2, interleukin-11, alpha-1-antitrypsin, antibodies or antibody fragments, and alternative protein scaffolds, in particular the protein is EPO, IFN alpha or G-CSF. Even more preferably, HAS 'is HES', and still more preferably HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, More preferably from about 5 to about 300 kDa, more preferably from about 10 to about 200 kDa, especially from about 50 to about 150 kDa, with molar substitution of from 0.1 to 3, preferably from 0.4 to 1.3, For example, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 or 1.3, preferably 0.7 to 1.3 such as 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, , And the C ₂ : C ₆ substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, even more preferably in the range of 3 to 12.

According to a particularly preferred embodiment, the present invention relates to a HAS derivative according to the formula:

In this formula, HAS is preferably HES, and more preferably HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, More preferably from about 5 to about 300 kDa, more preferably from about 10 to about 200 kDa, especially from about 50 to about 150 kDa, with molar substitution of from 0.1 to 3, preferably from 0.4 to 1.3, Wherein the C ₂ : C ₆ substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, more preferably in the range of 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, Preferably in the range of 3 to 12.

According to a further particularly preferred embodiment, the invention relates to a HAS derivative according to the formula:

In this formula, HAS is preferably HES, and more preferably HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, More preferably from about 5 to about 300 kDa, more preferably from about 10 to about 200 kDa, especially from about 50 to about 150 kDa, with molar substitution of from 0.1 to 3, preferably from 0.4 to 1.3, Wherein the C ₂ : C ₆ substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, more preferably in the range of 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, Preferably in the range of 3 to 12.

According to a further particularly preferred embodiment, the invention relates to a HAS derivative according to the formula:

In this formula, HAS is preferably HES, and more preferably HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, More preferably from about 5 to about 300 kDa, more preferably from about 10 to about 200 kDa, especially from about 50 to about 150 kDa, with molar substitution of from 0.1 to 3, preferably from 0.4 to 1.3, Wherein the C ₂ : C ₆ substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, more preferably in the range of 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, Preferably in the range of 3 to 12.

According to a further particularly preferred embodiment, the invention relates to a HAS derivative according to the formula:

In this formula, HAS is preferably HES, and more preferably HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, More preferably from about 5 to about 300 kDa, more preferably from about 10 to about 200 kDa, especially from about 50 to about 150 kDa, with molar substitution of from 0.1 to 3, preferably from 0.4 to 1.3, Wherein the C ₂ : C ₆ substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, more preferably in the range of 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, Preferably in the range of 3 to 12.

According to a further particularly preferred embodiment, the invention relates to a HAS derivative according to the formula:

In this formula, HAS is preferably HES, and more preferably HES has an average molecular weight of about 1 to about 1000 kDa, more preferably about 1 to about 800 kDa, more preferably about 1 to about 500 kDa, More preferably from about 5 to about 300 kDa, more preferably from about 10 to about 200 kDa, especially from about 50 to about 150 kDa, with molar substitution of from 0.1 to 3, preferably from 0.4 to 1.3, Wherein the C ₂ : C ₆ substitution ratio is preferably in the range of 2 to 20, more preferably in the range of 2 to 15, more preferably in the range of 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, Preferably in the range of 3 to 12.

According to a further aspect, the present invention relates to a HAS derivative comprising BA 'as mentioned above, for use in a method for the treatment of the human or animal body, or to a method as described above, obtainable or obtainable by a method as mentioned above Lt; RTI ID = 0.0 > BA ' as < / RTI >

Furthermore, the present invention relates to a HAS derivative comprising BA 'as mentioned above, as a therapeutic or prophylactic agent, or a HAS derivative comprising BA' as mentioned above, obtainable or obtainable by a method as mentioned above .

Furthermore, the present invention includes a therapeutically effective amount of a HAS derivative comprising BA 'as referred to above, or a HAS derivative comprising BA' as mentioned above, obtainable or obtainable by a method as mentioned above ≪ / RTI >

The HAS derivatives of the present invention, including BA ', can be administered by suitable methods, for example by the enteral, parenteral or pulmonary methods, which are preferably administered by intravenous, subcutaneous or intramuscular routes. The particular route chosen will depend on the disease to be treated. Preferably, the derivative is a suitable carrier as is known in the art (e. G., As used in first generation / unmodified biologics with or without albumin as an excipient), a suitable diluent, For example, with a sterile solution for intravenous, intramuscular or subcutaneous application. The dosage required will depend on the severity of the disease to be treated, the individual response of the patient, the mode of administration employed, and the like. A person skilled in the art can establish an accurate dosage based on his general knowledge.

As mentioned above, HAS derivatives may be used in combination with pharmaceutical excipients, so long as the pharmaceutical compositions according to the present invention comprising a HAS derivative containing BA 'are involved. In general, HAS derivatives will be in solid form, which can be combined with suitable pharmaceutical excipients, which may be in solid or liquid form. As excipients, mention may be made of carbohydrates, inorganic salts, antimicrobials, antioxidants, surfactants, buffers, acids, bases and combinations thereof. Carbohydrates such as sugars, derivatized sugars such as alditol, aldonic acid, esterified sugars and / or sugar polymers may be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; Disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; Polysaccharides such as raffinose, meletose, maltodextrin, dextran, starch and the like; And alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like. Excipients may also include inorganic salts or buffers, such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium monophosphate, sodium dihydrogenphosphate and combinations thereof. The pharmaceutical composition according to the present invention may also contain an antimicrobial agent for preventing or preventing microbial growth such as benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, Phenylmercuric acid nitrate, thimerosol, and combinations thereof. The pharmaceutical compositions according to the present invention may also be formulated as pharmaceutical compositions comprising an antioxidant such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium sulfite, Aldehyde sulfoxylate, sodium metabisulfite, and combinations thereof. The pharmaceutical composition according to the invention may also contain a surfactant such as polysorbate or pluronics sorbital esters; Lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamine acids and fatty esters; Steroids such as cholesterol; And chelating agents such as EDTA or zinc. The pharmaceutical compositions according to the present invention may also contain pharmaceutically acceptable excipients and / or diluents, such as, for example, hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, And / or may include sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, have. Generally, the excipient is present in the pharmaceutical composition according to the invention in an amount of 0.001 to 99.999 wt-%, preferably 0.01 to 99.99 wt-%, more preferably 0.1 to 99.9 wt-%, in each case based on the total weight of the pharmaceutical composition %. ≪ / RTI >

According to yet another aspect, the invention also relates to the use of a HAS derivative as defined above, preferably a HES derivative, wherein BA is EPO, for the manufacture of a medicament for the treatment of anemic disorders or hematopoietic disorders or diseases associated therewith ). ≪ / RTI >

According to yet another aspect, the present invention also relates to the use of a HAS derivative, preferably a HES derivative, as described above, for the manufacture of a medicament for the treatment of hemophilia A for the treatment of disorders characterized by hematopoietic or immune dysfunction Wherein BA is G-CSF. According to a preferred embodiment, the disorder characterized by hematopoiesis or immunodeficiency is a result of chemotherapy, radiotherapy, infectious disease, severe chronic neutropenia, or leukemia.

According to yet another aspect, the present invention also provides a method of treating leukemia, such as hair cell leukemia, chronic myelogenous leukemia, multiple myeloma, follicular lymphoma, cancer, such as carcinoid tumors, malignant melanoma and hepatitis , Preferably a HES derivative, wherein BA is IFN alpha, for the manufacture of a medicament for the treatment of, for example, chronic hepatitis B and chronic hepatitis C,

According to another aspect, the present invention also relates to the use of a HAS derivative as defined above, preferably a HES derivative, wherein BA is IFN gamma, for the manufacture of a medicament for the treatment of osteoporosis and / or chronic malignant diseases Lt; / RTI >

According to yet another aspect, the present invention also provides a pharmaceutical composition comprising a HAS derivative, preferably a HES derivative, wherein BA is IL-2, as described above, for the manufacture of a medicament for the treatment of osteoporosis and / .

According to yet another aspect, the present invention also relates to a HAS derivative as defined above, preferably a HES derivative, wherein BA is IL-11, for the manufacture of a medicament for the treatment of platelet transfusion following myelosuppressive chemotherapy ). ≪ / RTI >

According to yet another aspect, the present invention also relates to the use of a HAS derivative, preferably a HES derivative, as hereinbefore defined, for the manufacture of a medicament for the treatment of emphysema, cystic fibrosis, atopic dermatitis and / Quot; is < / RTI > A1AT).

According to yet another aspect, the present invention also relates to a pharmaceutical composition for the treatment of multiple sclerosis, preferably recurrent forms of multiple sclerosis, comprising a HAS derivative as defined above, preferably a HES derivative, wherein BA is an IFN beta Quot;). ≪ / RTI >

According to yet another aspect, the present invention also relates to a method for the treatment of a symptom manifestation in a hemophilia A or B patient with an inhibitor of Factor VIII or Factor IX, HES derivatives, wherein BA is a factor VII.

According to yet another aspect, the present invention also provides a method of controlling and preventing hemorrhagic symptom manifestations in a patient suffering from hemophilia B, such as, for example, congenital factor IX deficiency or Christmas disease, including controlling and preventing bleeding under surgical conditions. , Wherein the BA is a factor IX, for the manufacture of a medicament for the production of a medicament for the production of a medicament for the production of a medicament for the production of a medicament for the production of a medicament for the production of a medicament for the production of a medicament.

Bibliography

DESCRIPTION OF THE DRAWINGS [

Figure 1: SDS-PAGE analysis of the oxHES55 / 0.7-IFNa coupling reaction

1 shows an SDS-PAGE analysis of the oxHES-IFNa coupling reaction according to Example 2. Fig. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lanes 1-4: The reaction mixture according to example 2.

Successful HES conversion of the target protein (19 kDa) was visible as a smeary band representing a broad mass distribution ranging from 30 to> 200 kDa.

Figure 2: Anion exchange chromatography of the oxHES55 / 0.7-IFNa coupling reaction

Figure 2 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm on an ion exchange column of the oxHES55 / 0.7-IFNa coupling reaction according to Example 2. The chromatographic conditions are as follows:

Chromatography system: Akta Explorer 100 (supplied by GE Healthcare).

Column: Hi Trap Q HP 1 ml (supplied by GE Healthcare).

Solvent A: 10 mM Tris-Cl, pH 8.0.

Solvent B: 10 mM Tris-Cl, 0.5 M NaCl, pH 8.0.

Operating conditions: flow rate 1 ml / min, 21 ° C.

Performance parameters:

   Equilibrium 10 CV 0% B

   Sample load

   Cleaning 2 CV 0% B

   Elution 16 CV 0-50% B

   Playing 10 CV 100% B

   Rebalance 8 CV 0% B

Load: 2 mg protein / ml resin as reaction mixture according to Example 2, diluted 20-fold in eluent A and adjusted to pH 8.0.

An unreacted excess of HES is found in the perfusion. HES conversion attenuates the interaction between protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figure 3: SDS-PAGE analysis of the oxHES55 / 0.7-EPO coupling reaction

Figure 3 shows an SDS-PAGE analysis of the oxHES55 / 0.7-EPO coupling reaction according to Example 3. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: The reaction mixture according to Example 3.

Lane 2: EPO starting material prior to bonding.

Successful HES conversion of the target protein (about 35-40 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 55 to> 200 kDa.

Figure 4: Cation exchange chromatography of the oxHES55 / 0.7-EPO coupling reaction

Figure 4 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm on an ion exchange column of the oxHES55 / 0.7-EPO coupling reaction according to Example 3. The chromatographic conditions are as follows:

Chromatography system: Acta Explorer 100 (supplied by GE Healthcare).

Column: High Trap SP HP (supplied by GE Healthcare).

Solvent A: 20 mM sodium acetate, pH 4.0.

Solvent B: 20 mM sodium acetate, 1 M NaCl, pH 4.0.

Operating conditions: flow rate 5 ml / min, 21 ℃.

Performance parameters:

   Equilibrium 10 CV 0% B

   Sample load

   Cleaning 1 2 CV 0% B

   Cleaning 2 2 CV 10% B

   Elution 21 CV 10-52% B

   Playing 8 CV 100% B

   Rebalance 5 CV 0% B

Load: 2 mg protein / ml resin as the reaction mixture according to Example 3, diluted 10-fold in Solvent A.

An unreacted excess of HES is found in the perfusion. HES conversion attenuates the interaction between protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figure 5: SDS-PAGE analysis of the oxHES100 / 1.0-IFNa coupling reaction

5 shows an SDS-PAGE analysis of the oxHES-IFNa coupling reaction according to Example 5. Fig. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: The reaction mixture according to example 5.

Successful HES conversion of the target protein (19 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 50 to> 200 kDa.

Figure 6: Anion exchange chromatography of the oxHES100 / 1.0-IFNa coupling reaction

Figure 6 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm on an ion exchange column of the oxHES100 / 1.0-IFNa coupling reaction according to Example 5. The chromatographic conditions are as follows:

Chromatography system: Acta Explorer 100 (supplied by GE Healthcare).

Column: High Trap Q HP 5 ml (supplied by GE Healthcare).

Solvent A: 10 mM Tris-Cl, pH 8.0.

Solvent B: 10 mM Tris-Cl, 0.5 M NaCl, pH 8.0.

Operating conditions: flow rate 1 ml / min, 21 ° C.

Performance parameters:

   Equilibrium 10 CV 0% B

   Sample load

   Cleaning 2 CV 0% B

   Elution 12.5 CV 0-50% B

   Playback 5 CV 100% B

   Rebalance 5 CV 0% B

Load: 2 mg protein / ml resin as reaction mixture according to Example 5, diluted 20 times in eluent A and adjusted to pH 8.0.

An unreacted excess of HES is found in the perfusion. HES conversion attenuates the interaction between protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figure 7: Peptide mapping of oxHES100 / 1.0-IFNa conjugates

Figure 7 shows the chromatographic separation of the IEX-purified oxHES-IFNa conjugate according to Example 5 treated with endo-LysC.

Proteolysis was performed using 7.5% endo-LysC in 50 mM Tris-Cl, pH 8.6, 0.01% SDS overnight at 37 ° C. The sample was denatured using DTT and guanidium chloride and analyzed by RP-HPLC on a 4.6 x 250 mm Jupiter C4 column (Phenomenex) performed with a water / acetonitrile gradient followed by TFA . The chromatogram shown was monitored at 214 nm.

The arrows show a visible chromatogram area between the chromatogram for protein (A) and the chromatogram for conjugate (B). Peaks for L1 and L1 / L2 (N-terminal peptides generated from endo-Lys C treatment) are significantly reduced for conjugate samples while other fragments are scarcely affected. These data suggest that HES is preferentially coupled to the N-terminus of IFNa.

Figure 8: SDS-PAGE analysis of the oxHES100 / 1.0-EPO coupling reaction

FIG. 8 shows an SDS-PAGE analysis of the oxHES100 / 1.0-EPO coupling reaction according to Example 6. FIG. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: 10 [mu] g EPO starting material prior to conjugation.

Lane 2: 5 EP EPO starting material prior to conjugation.

Lane 3: Reaction mixture according to example 6.

Successful HES conversion of the target protein (about 35-40 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 70 to> 200 kDa.

Figure 9: Cation exchange chromatography of the oxHES100 / 1.0-EPO coupling reaction

Figure 9 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm on an ion exchange column of the oxHESlOO / 1.0-EPO coupling reaction according to Example 6. The chromatographic conditions are as follows:

Chromatography system: Acta Explorer 100 (supplied by GE Healthcare).

Column: 2 x 5 ml HiTrap SP HP (supplied by GE Healthcare).

Solvent A: 20 mM sodium acetate, pH 4.0.

Solvent B: 20 mM sodium acetate, 1 M NaCl, pH 4.0.

Operating conditions: flow rate 5 ml / min, 21 ℃.

Performance parameters:

   Equilibrium 10 CV 0% B

   Sample load

   Cleaning 1 2 CV 0% B

   Cleaning 2 2 CV 10% B

   Elution 21 CV 10-52% B

   Playback 2.5 CV 100% B

   Rebalance 5 CV 0% B

Load: 2 mg protein / ml resin as reaction mixture according to Example 6, diluted 10-fold in eluent A.

An unreacted excess of HES is found in the perfusion. HES conversion attenuates the interaction between protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figure 10: SDS-PAGE analysis of the oxHES100 / 1.0-G-CSF coupling reaction

10 shows an SDS-PAGE analysis of the oxHES100 / 1.0-G-CSF coupling reaction according to Example 7. Fig. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: The reaction mixture according to example 7.

Lane 2: G-CSF starting material prior to conjugation.

Successful HES conversion of the target protein (approximately 18 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 50 to> 200 kDa.

Figure 11: RP-HPLC analysis of the oxHES100 / 1.0-G-CSF coupling reaction

Figure 11 shows an RP-HPLC analysis flake of the oxHES100 / 1.0-G-CSF coupling reaction according to Example 7 monitored by UV-Vis spectroscopy at 221 nm. The chromatographic conditions are as follows:

Chromatography system: Summit, P580 (HPG) (supplier: Dionex).

Column: Jupiter C18, 300A, 5 占 퐉, 4.6 占 150 mm (supplied by Phenomenex).

Solvent A: 0.1% trifluoroacetic acid in water.

Solvent B: 0.1% trifluoroacetic acid in acetonitrile.

Operating conditions: flow rate 1 ml / min, 20 ° C.

Gradient: 0-5 minutes, 5-55% B; 5-12 minutes, 55-68% B; 12-17 min, 100% B; 17-22 min, 5% B; Draft delay 2.5 minutes.

Load: 10 μg protein as a reaction mixture diluted in water to a protein concentration of 0.1 mg / ml.

The main peak at 11.5 min is a HES protein conjugate isolated from free G-CSF eluting at about 13 min.

Figure 12: SDS-PAGE analysis of HES100 / 1.0-IFNa coupling reaction

12 shows an SDS-PAGE analysis of the HES-IFNa coupling reaction according to Example 10. Fig. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, unstained protein marker 5 to 200 kDa (source: Serva).

Lane 1: The reaction mixture according to Example 9.

Successful HES conversion of the target protein (19 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 50 to> 200 kDa.

Figure 13: Anion exchange chromatography of HES100 / 1.0-IFNa coupling reaction

Figure 13 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm on an ion exchange column of the HES 100 / 1.0-IFNa coupling reaction according to Example 10. The chromatographic conditions are as follows:

Chromatography system: Acta Explorer 100 (supplied by GE Healthcare).

Column: 5 ml HiTrap SP HP (supplied by GE Healthcare).

Solvent A: 20 mM sodium acetate, pH 4.0.

Solvent B: 20 mM sodium acetate, 1 M NaCl, pH 4.0.

Operating conditions: flow rate 5 ml / min, 21 ℃.

Performance parameters:

   Equilibrium 10 CV 0% B

   Sample load

   Cleaning 1 2 CV 0% B

   Elution 20 CV 0-50% B

   Playing 10 CV 100% B

   Rebalance 5 CV 0% B

Load: 3 mg protein / ml resin as reaction mixture according to Example 9, diluted 10-fold in eluent A.

An unreacted excess of HES is found in the perfusion. HES conversion attenuates the interaction between protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figure 14: SDS-PAGE analysis of HES100 / 1.0-EPO coupling reaction

14 shows an SDS-PAGE analysis of the HES100 / 1.0-EPO coupling reaction according to Example 11. Fig. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: IEX purified HES EPO conjugate.

Lane 2: The reaction mixture according to example 11.

Lane 3: 5 EP EPO starting material prior to conjugation.

Successful HES conversion of the target protein (about 35-40 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 70 to> 200 kDa.

Figure 15: Cation exchange chromatography of HES100 / 1.0-EPO coupling reaction

Figure 15 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm on an ion exchange column of a HES 100 / 1.0-EPO coupling reaction according to Example 11. The chromatographic conditions are as follows:

Chromatography system: Acta Explorer 100 (supplied by GE Healthcare).

Column: 4 x 5 ml HiTrap SP HP (supplied by GE Healthcare).

Solvent A: 20 mM sodium acetate, pH 4.0.

Solvent B: 20 mM sodium acetate, 1 M NaCl, pH 4.0.

Operating conditions: flow rate 5 ml / min, 21 ℃.

Performance parameters:

   Equilibrium 5 CV 0% B

   Sample load

   Cleaning 1 2 CV 0% B

   Elution 13 CV 0-52% B

   Playback 2.5 CV 100% B

   Rebalance 2.5 CV 0% B

Load: 3 mg protein / ml resin as reaction mixture according to Example 11, diluted twice in eluent A.

An unreacted excess of HES is found in the perfusion. HES conversion attenuates the interaction between protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figure 16: SDS-PAGE analysis of HES100 / 1.0-G-CSF coupling reaction

16 shows an SDS-PAGE analysis of the HES100 / 1.0-G-CSF coupling reaction according to Example 12. Fig. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: IEX purified HES G-CSF conjugate.

Lane 2: The reaction mixture according to example 12.

Lane 3: 5 [mu] g G-CSF starting material prior to conjugation.

Successful HES conversion of the target protein (approximately 18 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 50 to> 200 kDa.

Figure 17: RP-HPLC analysis of HES100 / 1.0-G-CSF coupling reaction

Figure 17 shows an RP-HPLC analysis flake of a HES100 / 1.0-G-CSF coupling reaction according to Example 12 monitored by UV-Vis spectroscopy at 221 nm. The chromatographic conditions are as follows:

Chromatography system: Sumit, P580 (LPG) (supplier: Dionex).

Column: Jupiter C18, 300A, 5 占 퐉, 4.6 占 150 mm (supplied by Phenomenex).

Solvent A: 0.1% trifluoroacetic acid in water.

Solvent B: 0.1% trifluoroacetic acid in acetonitrile.

Operating conditions: flow rate 1 ml / min, 20 ° C.

Gradient: 0-5 minutes, 5-55% B; 5-12 minutes, 55-68% B; 12-17 min, 100% B; 17-22 min, 5% B; Draft delay 2.5 minutes.

Load: 10 μg protein as a reaction mixture diluted in water to a protein concentration of 0.1 mg / ml.

The main peak at 10 to 10.5 min is a HES protein conjugate isolated from free G-CSF eluting at about 12 min.

Figure 18: Cation exchange chromatography of HES100 / 1.0-G-CSF coupling reaction

Figure 18 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm on an ion exchange column of a HES 100 / 1.0-G-CSF coupling reaction according to Example 12. The chromatographic conditions are as follows:

Chromatography system: Acta Explorer 100 (supplied by GE Healthcare).

Column: 2 x 5 ml HiTrap SP HP (supplied by GE Healthcare).

Solvent A: 20 mM sodium acetate, pH 4.0.

Solvent B: 20 mM sodium acetate, 1 M NaCl, pH 4.0.

Operating conditions: flow rate 5 ml / min, 21 ℃.

Performance parameters:

   Equilibrium 5 CV 0% B

   Sample load

   Cleaning 2 CV 0% B

   Elution 15 CV 0-40% B

   Playback 5 CV 100% B

   Rebalance 5 CV 0% B

Load: 3 mg protein / ml resin as reaction mixture according to Example 12, diluted twice in eluent A.

An unreacted excess of HES is found in the perfusion. HES conversion attenuates the interaction between protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figures 19-23 are referred to in the context of the respective embodiments.

24

Figure 24 shows an HPGPC analysis flake of an oxHBS-BSA coupling reaction according to "Additional Data (A.2) " monitored by UV-Vis spectroscopy at 280 nm. The chromatographic conditions are as follows:

Chromatography system: Shimadsu LC1O AD / UV-Detektor: TSP UV 2000.

Column: Superose 6 10/300 GL (supplied by Pharmacia).

Solvent: Phosphate Buffer: [3.887 g Na ₂ HPO ₄ x 2 H ₂ O, 1.967 g NaH ₂ PO ₄ x 2 H ₂ O, 11.688 g NaCl, 0.05 g NaN ₃ to a total volume of 1.0 L or less Reagent Pharmakopoea Europaea). The solution was filtered using a 0.45 [mu] m filter.

Operating conditions: flow rate 0.4 ml / min, 20 ° C.

Load: 0.9 mg protein as a reaction mixture, dissolved in 100 μl to a protein concentration of 9 mg / ml.

The upper part shows the BSA starting material before the coupling reaction. The peaks from left to right are below 38.038, 39.277, and 42.272.

The lower part shows the HBS-BSA conjugate. The peaks from left to right are below 36.795, 39.345, and 41.521.

Figure 25: SDS-PAGE analysis of oxHBS-IFNa coupling reaction

Figure 25 shows an SDS-PAGE analysis of the oxHBS-IFNa coupling reaction according to "Additional Data (A.3) ". Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lanes 1 to 4: reaction mixture according to "additional data (A.3) ".

Successful HBS conversion of the target protein (about 19 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 30 to> 200 kDa.

Figure 26: Anion exchange chromatography of the oxHBS-IFNa coupling reaction

Figure 26 shows the chromatographic separation monitored by UV-Vis spectroscopy at 221 nm of the oxHBS-IFNa coupling reaction according to the additional data (A.3) using an ion exchange column. The chromatographic conditions are as follows:

Chromatography system: Acta Explorer 100 (supplied by GE Healthcare).

Column: HiTrap Q HP 1 ml (supplied by GE Healthcare).

Solvent A: 10 mM Tris-Cl, pH 8.0.

Solvent B: 10 mM Tris-Cl, 0.5 M NaCl, pH 8.0.

Operating conditions: flow rate 1 ml / min, 20 ° C.

Performance parameters:

   Equilibrium 10 CV 0% B

   Sample load

   Cleaning 2 CV 0% B

   Elution 16 CV 0-50% B

   Playing 10 CV 100% B

   Rebalance 8 CV 0% B

Load: The reaction mixture according to Example 3, diluted 20-fold in eluent A and adjusted to pH 8.0.

An unreacted excess of HBS is found in the perfusion. HBS formation weakens the interaction of protein and column, reducing the elution time for the conjugate compared to unmodified protein.

Figure 27: SDS-PAGE analysis of oxHBS-EPO coupling reaction

Figure 27 shows an SDS-PAGE analysis of the oxHBS-EPO coupling reaction according to "Additional Data (A.4) ". Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions.

Load: 10 [mu] g protein as reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: reaction mixture according to "additional data (A.4) ".

Lane 2: EPO starting material prior to bonding.

Successful HBS conversion of the target protein (about 35-40 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 45 to> 200 kDa.

Figure 28: HESlOO / 1.0, linker (a2) and (Trp ⁴ ) - RP-HPLC analysis of the coupling reaction using Kempted

Figure 28 shows RP-HPLC analysis flakes of the coupling reaction with HESlOO / 1.0, linker (a2) and (Trp ⁴ ) -matched according to Example 18, Table 3 monitored by UV-Vis spectroscopy at 221 nm Respectively. The chromatographic conditions are as follows:

Chromatography system: Shimadu LC 20 Prominence, LC 20AT (LPG) (Shimadzu)

Column: Jupiter C18, 300A, 5 占 퐉, 4.6 占 150 mm (supplied by Phenomenex).

Solvent A: 0.1% trifluoroacetic acid in water.

Solvent B: 0.1% trifluoroacetic acid in acetonitrile.

Operating conditions: flow rate 1 ml / min, 20 ° C.

Gradient: 0-15 minutes, 2-30% B; 15-20 minutes, 30-98% B; 20-27 minutes, 2% B.

Load: 5 μg protein as a reaction mixture, diluted in water to a protein concentration of 0.05 mg / ml.

The main peak at 11.5 to 15 min is the HES peptide conjugate isolated from the free (Trp ⁴ ) -memtide eluting at about 17 min.

29: HESlOO / 1.0, linkers (a16) and (Trp ⁴ ) - RP-HPLC analysis of the coupling reaction using Kempted

Figure 29 shows the RP-HPLC of the coupling reaction with HESlOO / 1.0, linker (a16) and (Trp ⁴ ) -memtpt according to Example 18, Table 5, line 13 monitored by UV-Vis spectroscopy at 221 nm It shows the analysis slice. The chromatographic conditions are as follows:

Chromatography system: Shimadzu LC 20 Prominence, LC 20AT (LPG) (Shimadzu)

Column: Jupiter C18, 300A, 5 占 퐉, 4.6 占 150 mm (supplied by Phenomenex).

Solvent A: 0.1% trifluoroacetic acid in water.

Solvent B: 0.1% trifluoroacetic acid in acetonitrile.

Operating conditions: flow rate 1 ml / min, 20 ° C.

Gradient: 0-15 minutes, 2-30% B; 15-20 minutes, 30-98% B; 20-27 minutes, 2% B.

Load: 5 μg protein as a reaction mixture, diluted in water to a protein concentration of 0.05 mg / ml.

The main peak at 11.5 to 15 min is the HES peptide conjugate isolated from the free (Trp ⁴ ) -memtide eluting at about 17 min.

30: HESlOO / 1.0, linkers (a11) and (Trp ⁴ ) - RP-HPLC analysis of the coupling reaction using Kempted

Figure 30 shows the RP-HPLC of the coupling reaction with HESlOO / 1.0, linker (a11) and (Trp ⁴ ) -matched according to Example 18, Table 5, line 24 monitored by UV-Vis spectroscopy under 221 nm It shows the analysis slice. The chromatographic conditions are as follows:

Chromatography system: Shimadzu LC 20 Prominence, LC 20AT (LPG) (Shimadzu)

Column: Jupiter C18, 300A, 5 占 퐉, 4.6 占 150 mm (supplied by Phenomenex).

Solvent A: 0.1% trifluoroacetic acid in water.

Solvent B: 0.1% trifluoroacetic acid in acetonitrile.

Operating conditions: flow rate 1 ml / min, 20 ° C.

Gradient: 0-15 minutes, 2-30% B; 15-20 minutes, 30-98% B; 20-27 minutes, 2% B.

Load: 5 μg protein as a reaction mixture, diluted in water to a protein concentration of 0.05 mg / ml.

The main peak at 11.5 to 15 min is the HES peptide conjugate isolated from the free (Trp ⁴ ) -memtide eluting at about 17 min.

31: HESlOO / 1.0, linker (a12) and (Trp ⁴ ) - RP-HPLC analysis of the coupling reaction using Kempted

31 shows the RP-HPLC of the coupling reaction with HESlOO / 1.0, linker (a12) and (Trp ⁴ ) -memtpt according to Example 18, Table 5, line 30 monitored by UV-Vis spectroscopy under 221 nm It shows the analysis slice. The chromatographic conditions are as follows:

Chromatography system: Shimadzu LC 20 Prominence, LC 20AT (LPG) (Shimadzu)

Column: Jupiter C18, 300A, 5 占 퐉, 4.6 占 150 mm (supplied by Phenomenex).

Solvent A: 0.1% trifluoroacetic acid in water.

Solvent B: 0.1% trifluoroacetic acid in acetonitrile.

Operating conditions: flow rate 1 ml / min, 20 ° C.

Gradient: 0-15 minutes, 2-30% B; 15-20 minutes, 30-98% B; 20-27 minutes, 2% B.

Load: 5 μg protein as a reaction mixture, diluted in water to a protein concentration of 0.05 mg / ml.

The main peak at 11.5 to 15 min is the HES peptide conjugate isolated from the free (Trp ⁴ ) -memtide eluting at about 17 min.

Figure 32: SDS-PAGE analysis of HES100 / 1.0-G-CSF coupling reaction

32 shows an SDS-PAGE analysis of the HES100 / 1.0-G-CSF coupling reaction according to Example 18. FIG. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MES performance buffer according to the manufacturer's instructions.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: The reaction mixture according to Table 5, line 7 (loaded with 10 [mu] g protein).

Lane 2: The reaction mixture according to Table 5, line 16 (loaded with 10 [mu] g protein).

Lane 3: The reaction mixture according to Table 5, line 27 (loaded with 10 [mu] g protein).

Lane 4: The reaction mixture according to Table 5, line 33 (10 ug protein loaded).

Lane 5: 0.5 [mu] g rhG-CSF starting material prior to conjugation.

Successful HES conversion of the target protein (approximately 18 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 50 to> 200 kDa.

Figure 33: SDS-PAGE analysis of HES100 / 1.0-IFNa and HES100 / 1.0-EPO coupling reactions

33 shows an SDS-PAGE analysis of the coupling reaction between HES100 / 1.0 and rhIFN? Or rhEPO according to Example 18. FIG. Separation was performed under reducing conditions using a NuPAGE system (Invitrogen) with 4-12% Bis-Tris gel (1.0 mm) and MOPS performance buffer according to the manufacturer's instructions. In the case of electrophoretic separation shown on the right, MES buffer was used instead of MOPS.

Typical load: 10 ug protein as starting material or reaction mixture.

M: Marker, Mark 12 (supplied by Invitrogen).

Lane 1: The reaction mixture according to Table 5, line 4.

Lane 2: The reaction mixture according to Table 5, line 14.

Lane 3: The reaction mixture according to Table 5, line 25.

Lane 4: The reaction mixture according to Table 5, line 31.

Lane 5: rhIFNα starting material prior to conjugation.

Successful HES conversion of the target protein (about 19 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 50 to> 200 kDa.

Lane 6: The reaction mixture according to Table 5, line 15.

Lane 7: The reaction mixture according to Table 5, line 5.

Lane 8: The reaction mixture according to Table 5, line 32.

Lane 9: rhEPO starting material prior to bonding.

Lane 10: The reaction mixture according to Table 5, line 26.

Successful HES conversion of the target protein (about 35-40 kDa) was visible as a stained band representing a wide range of mass distributions ranging from 60 to> 200 kDa.

<Examples>

Example 1: Preparation of oxHES55 / 0.7-N- (3-propioldehyde diethyl acetal)

HES aldonic acid (oxHES) was synthesized starting from HES (HES55 / 0.7) with a molecular weight of 55 kDa and a molar substitution of 0.7 as described in Example 9 of WO 2005/083103 A &Lt; / RTI &gt; is described).

3 O g oxHES 55 / 0.7 dried for 2 days at 80 ° C was dissolved in 60 ml anhydrous dimethylformamide (DMF) and the solution was heated to 70 ° C. 25 g of 1-amino-3,3-diethoxypropane in 50 ml of anhydrous DMF was added and the reaction mixture was heated at 70 DEG C for 48 hours.

A rotary evaporator was used to remove DMF and excess 1-amino-3, 3-diethoxypropane under vacuum at 60-80 &lt; 0 &gt; C. The remaining crude solid was washed with acetone until no color was detected in the wash solution. The product was dissolved in 500 ml water and purified by ultrafiltration using a membrane with a cut-off of 10,000 Daltons. When the pH of the preservative reached 6 to 7, it was readjusted to 9 using 0.1 M sodium hydroxide solution. This process was repeated four times. Finally, the product was lyophilized.

Example 2: Preparation of oxHES55 / 0.7 interferon alpha 2b (IFNa) conjugate

A suitable amount of 10 mM HCl was added to 400 mg of acetal prepared in Example 1 to give a solution having a concentration of 40% (w / v). This solution was incubated at 21 占 폚 for 24 hours under stirring to deprotect the aldehyde functional group. The pH value was adjusted to the value used in the conjugation buffer by adding 0.1 M NaOH.

Recombinant human interferon alpha-2b produced by recombinant DNA technology with interferon-alpha [ Escherichia coli ( E. coli ); Such interferon alpha-2b is composed of 165 amino acids and presents the same amino acid sequence as natural human interferon alpha-2b (hIFN-alpha-2b)] is concentrated to 16 mg / ml or less, And transferred to a suitable conjugation buffer (0.1 M sodium acetate buffer, pH 4.0).

A 10-fold molar excess of oxHES aldehyde (based on M _w ) was used with a protein concentration of 6 mg / ml in the reaction mixture; The concentration of oxHES aldehyde was 20% (w / v). The deprotected oxHES aldehyde was combined with the protein solution and the reductive amination reaction was started by adding freshly prepared NaCNBH ₃ solution (0.5 M in conjugation buffer) to obtain a reducing agent with a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 10 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 1) and by reversed phase chromatography on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the conjugation yield. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

The HES-interferon-alpha was isolated from the unreacted compound by anion exchange chromatography using a Q HP column on an Acta system (GE Healthcare). The eluent A was 10 mM Tris-Cl, pH 8.0, and the eluent B was 10 mM Tris-Cl, 0.5 M NaCl, pH 8.0. The gradient for separating the conjugate from unmodified protein was 0% B => 50% B in 16 CV (FIG. 2).

Example 3: Preparation of oxHES55 / 0.7 erythropoietin (EPO) conjugate

A suitable amount of 10 mM HCl was added to 400 mg of acetal prepared in Example 1 to give a solution having a concentration of 40% (w / v). This solution was incubated at 21 占 폚 for 24 hours under stirring to deprotect the aldehyde functional group. The pH value was adjusted to the value used in the conjugation buffer by adding 0.1 M NaOH.

The deprotected oxHES aldehyde was dissolved in EPO [recombinant human EPO] solution having amino acid sequence of human EPO and having almost the same characteristics as commercially available Erypo® (Ortho Biotech, Jansen-Cilag) or NeoRecormon® (supplied by Roche) 10 mg / ml in reaction buffer, 0.1 M sodium acetate buffer, pH 5). The OxHES aldehyde was added in a 10-fold molar excess (based on M _w ) compared to the EPO concentration. The resulting reaction mixture had an EPO concentration of 5 mg / ml and an oxHES aldehyde concentration of 10% (w / v). Reductive amination reaction was started by adding 0.5 M NaCNBH ₃ solution constituted in the reaction buffer to obtain a reducing agent having a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 0 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 3) and by reversed phase chromatography on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the yield of conjugation. Elution of the RP-HPLC was performed using an acid water / acetonitrile gradient with 0.1% TFA.

The HESylated EPO was isolated from unreacted compounds by cation exchange chromatography using an SP HP column on an Acta system (GE Healthcare). The eluent A was 20 mM sodium acetate, pH 4.0, and the eluent B was 20 mM sodium acetate, 1 M NaCl, pH 4.0. The gradient for separating the conjugate and unmodified protein was 10% B, 2 CV; 10% B => 52% B in 21 CV (FIG. 4).

The HES-coupled region in the target protein was identified by peptide mapping the IEX-purified HES-protein conjugate. The conjugate was digested (2% endoproteinase Lys-C, pH 8.6, 37 [deg.] C, overnight) with the appropriate protease and the resulting fragment was purified using an acidic water / acetonitrile gradient with TFA C4 column (supplied by Phenomenex, Jupiter). The site of HES conversion in the protein was indirectly confirmed by the reduction or disappearance of each peptide in the chromatogram as compared to the control digest of the target protein alone.

Example 4: Preparation of oxHES100 / 1.0-N- (3-propioldehyde diethyl acetal)

HES aldonic acid (oxHES) was synthesized starting from HES (HES100 / 1.0) with a molecular weight of 100 kDa and a molar substitution of 1.0 as described in Example 9 of WO 2005/083103 A &Lt; / RTI &gt; is described).

3 O g oxHES 100 / 1.0 dried for 2 days at 80 ° C was dissolved in 150 ml anhydrous dimethylformamide (DMF) and the solution was heated to 70 ° C. 25 g of 1-amino-3,3-diethoxypropane in 60 ml of anhydrous DMF was added and the reaction mixture was heated at 70 DEG C for 48 hours.

A rotary evaporator was used to remove DMF and excess 1-amino-3, 3-diethoxypropane under vacuum at 60-80 &lt; 0 &gt; C. The remaining crude solid was washed with acetone until no color was detected in the wash solution. The product was dissolved in 500 ml water and purified by ultrafiltration using a membrane with a cut-off of 10,000 Daltons. When the pH of the preservative reached 6 to 7, it was readjusted to 9 using 0.1 M sodium hydroxide solution. This process was repeated four times. Finally, the product was lyophilized.

Example 5: Preparation of HES100 / 1.0 interferon alpha (IFNa) conjugate from oxidized HES

A suitable amount of 10 mM HCl was added to 400 mg of the acetal prepared in Example 4 to give a solution having a concentration of 40% (w / v). The solution was incubated overnight at &lt; RTI ID = 0.0 &gt; 21 C &lt; / RTI &gt; with stirring to deprotect the aldehyde functionality. The pH value was adjusted to the value used in the conjugation buffer by adding 0.1 M NaOH.

Recombinant human interferon alpha-2b produced by recombinant DNA technology using interferon-alpha [Escherichia coli (E. coli)]; Such interferon alpha-2b is composed of 165 amino acids and presents the same amino acid sequence as natural human interferon alpha-2b (hIFN-alpha-2b)] is concentrated to 16 mg / ml or less, And transferred to a suitable conjugation buffer (0.1 M sodium acetate buffer, pH 4.0).

A 6-fold molar excess of oxHES aldehyde (based on M _n ) was used with a final protein concentration of 8 mg / ml in the reaction mixture; The concentration of oxHES aldehyde was 20% (w / v). The deprotected oxHES aldehyde was combined with the protein solution and the reductive amination reaction was started by adding freshly prepared NaCNBH ₃ solution (0.5 M in conjugation buffer) to obtain a reducing agent with a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 5 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 5) and by reverse phase chromatography on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the yield of conjugation. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

The HES-interferon-alpha was isolated from the unreacted compound by anion exchange chromatography using a Q HP column on an Acta system (GE Healthcare). The eluent A was 10 mM Tris-Cl, pH 8.0, and the eluent B was 10 mM Tris-Cl, 0.5 M NaCl, pH 8.0. The gradient for separating the conjugate from unmodified protein was 0% B => 50% B in 12.5 CV (FIG. 6).

The HES-coupled region in the target protein was identified by peptide mapping the IEX-purified HES-protein conjugate. The conjugate was digested (2% endoproteinase Lys-C, pH 8.6, 37 [deg.] C, overnight) with the appropriate protease and the resulting fragment was purified using an acidic water / acetonitrile gradient with TFA C4 column (supplied by Phenomenex, Jupiter). The site of HES conversion in the protein was indirectly confirmed by the reduction or disappearance of each peptide in the chromatogram as compared to the control digest of the target protein alone.

Example 6: Preparation of HES100 / 1.0 erythropoietin (EPO) conjugate from oxidized HES

A suitable amount of 10 mM HCl was added to 400 mg of the acetal prepared in Example 4 to give a solution having a concentration of 40% (w / v). The solution was incubated at 21 DEG C for 24 hours to deprotect the aldehyde functional group. The pH value was adjusted to the value used in the conjugation buffer by adding 0.1 M NaOH.

The deprotected oxHES aldehyde was dissolved in EPO [recombinant human EPO] solution having amino acid sequence of human EPO and having almost the same characteristics as commercially available Erypo® (Ortho Biotech, Jansen-Cilag) or NeoRecormon® (supplied by Roche) 10 mg / ml in reaction buffer, 0.1 M sodium acetate buffer, pH 5). The OxHES aldehyde was added in a 15-fold molar excess (based on M _n ) compared to the EPO concentration. The resulting reaction mixture had an EPO concentration of 3.7 mg / ml and an oxHES aldehyde concentration of 15% (w / v). Reductive amination reaction was started by adding 0.5 M NaCNBH ₃ solution constituted in the reaction buffer to obtain a reducing agent having a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 10 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 8) and reverse phase chromatography on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the yield of conjugation. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

The HESylated EPO was isolated from unreacted compounds by cation exchange chromatography using an SP HP column on an Acta system (GE Healthcare). The eluent A was 20 mM sodium acetate, pH 4.0, and the eluent B was 20 mM sodium acetate, 1 M NaCl, pH 4.0. The gradient for separating the conjugate and unmodified protein was 10% B, 2 CV; 10% B => 52% B in 21 CV (FIG. 9).

The HES-coupled region in the target protein was identified by peptide mapping the IEX-purified HES-protein conjugate. The conjugate was digested (2% endoproteinase Lys-C, pH 8.6, 37 [deg.] C, overnight) with the appropriate protease and the resulting fragment was purified using an acidic water / acetonitrile gradient with TFA C4 column (supplied by Phenomenex, Jupiter). The site of HES conversion in the protein was indirectly confirmed by the reduction or disappearance of each peptide in the chromatogram as compared to the control digest of the target protein alone.

Example 7: Preparation of HES100 / 1.0 granulocyte colony stimulating factor (G-CSF) conjugate from oxidized HES

A suitable amount of 10 mM HCl was added to 400 mg of the acetal prepared in Example 4 to give a solution having a concentration of 40% (w / v). The solution was incubated overnight at &lt; RTI ID = 0.0 &gt; 21 C &lt; / RTI &gt; to deprotect the aldehyde functionality. The pH value was adjusted to the value used in the conjugation buffer by adding 0.1 M NaOH.

The deprotected oxHES aldehyde was dissolved in rh-Met-G-CSF solution [reaction buffer 5 mg / ml in 0.1 M sodium acetate buffer, pH 5; It has nearly the same amino acid sequence as the commercially available Neupogen® (supplier: Amgen, Munchen, D). G-CSF expressed by cola]. OxHES aldehyde was added at a 30-fold molar excess (based on M _n ) compared to the G-CSF concentration. The G-CSF concentration in the resulting reaction mixture was 1.9 mg / ml and the oxHES aldehyde concentration was 20% (w / v). Reductive amination reaction was started by adding 0.5 M NaCNBH ₃ solution constituted in the reaction buffer to obtain a reducing agent having a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 0 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 10) and reverse phase chromatography (Figure 11) on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the yield of conjugation. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

Example 8: Preparation of HES100 / 1.0-N- (3-propioldehyde diethyl acetal)

15 g of HES 100 / 1.0 was dissolved in 35 g of sodium acetate buffer (pH = 5 and c = 1 mol / l) and 2.07 ml of 1-amino-3,3-diethoxypropane as well as 1.885 g of sodium cyanobor Was added. The reaction mixture was stirred at 60 ° C for 16-24 hours, diluted with 100 ml water, neutralized with dilute sodium hydroxide solution and treated with ammonium bicarbonate buffer (pH = 9, c = 10 mmol / 1, 45 cycles) But was post-treated by ultrafiltration using a membrane with a cut-off of 10,000 Da against water (for the last 5 exchange cycles). The purified and concentrated HES derivative solution (approximately 20 wt-%) was dialyzed against a sodium hydroxide solution (pH = 12) at 60 ° C using a membrane with a cut-off of 10,000 Da. The product was then isolated by lyophilization.

Example 9: Preparation of HES100 / 1.0-N- (3-propionaldehyde)

10 g of HES-N- (3-propionaldehyde diethyl acetal) from Example 8 is dissolved in 100 ml of aqueous HCl, pH = 2 (c = 10 mmol / l) &Lt; / RTI &gt; The reaction mixture was purified by ultrafiltration using a membrane with a cut-off of 10,000 Da against aqueous HCl, pH = 2 (10 cycles) as well as water (for the last 5 wash cycles). Isolation of the product was performed by lyophilization.

Example 10: Preparation of HES100 / 1.0 interferon alpha (IFNa) conjugate from HES

A suitable amount of 10 mM HCl was added to 400 mg of acetal prepared in Example 8 to give a solution having a concentration of 40% (w / v). The solution was incubated overnight at &lt; RTI ID = 0.0 &gt; 21 C &lt; / RTI &gt; with stirring to deprotect the aldehyde functionality. The pH value was adjusted to the value used in the conjugation buffer by adding 0.1 M NaOH.

Recombinant human interferon alpha-2b produced by recombinant DNA technology using interferon-alpha [Escherichia coli (E. coli)]; Such interferon alpha-2b is composed of 165 amino acids and presents the same amino acid sequence as natural human interferon alpha-2b (hIFN-alpha-2b)] is concentrated to 16 mg / ml or less, And transferred to a suitable conjugation buffer (0.1 M sodium acetate buffer, pH 4.0).

A 5-fold molar excess of HES aldehyde (based on M _w ) was used with a final protein concentration of 7 mg / ml in the reaction mixture; The HES aldehyde concentration was 18% (w / v). The deprotected HES aldehyde was combined with the protein solution and a reductive amination reaction was initiated by adding freshly prepared NaCNBH ₃ solution (0.5 M in conjugation buffer) to give a reducing agent with a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 5 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 12) and reverse phase chromatography on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the yield of conjugation. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

The HES-IFN alpha was isolated from unreacted compounds by cation exchange chromatography using an SP HP column on an Akta system (GE Healthcare). The eluent A was 20 mM sodium acetate, pH 4.0, and the eluent B was 20 mM sodium acetate, 1 M NaCl, pH 4.0. The gradient for separating the conjugate from unmodified protein was 0% B => 50% B in 20 CV (FIG. 13).

The HES-coupled region in the target protein was identified by peptide mapping the IEX-purified HES-protein conjugate. The conjugate was digested (2% endoproteinase Lys-C, pH 8.6, 37 [deg.] C, overnight) with the appropriate protease and the resulting fragment was purified using an acidic water / acetonitrile gradient with TFA C4 column (supplied by Phenomenex, Jupiter). The site of HES conversion in the protein was indirectly confirmed by the reduction or disappearance of each peptide in the chromatogram as compared to the control digest of the target protein alone.

Example 11: Preparation of HES100 / 1.0 erythropoietin (EPO) conjugate from HES

The deprotected HES aldehyde from Example 9 was reacted with EPO [recombinant protein having amino acid sequence of human EPO and almost the same characteristics as commercially available Erypo® (Ortho Biotech, Jansen-Cilag) or NeoRecormon® (source: Roche) Human EPO] solution (10 mg / ml in reaction buffer 0.1 M sodium acetate buffer, pH 5). The HES aldehyde was added in a 40-fold molar excess (based on M _n ) compared to the EPO concentration. The resulting reaction mixture had an EPO concentration of 3.2 mg / ml and a HES aldehyde concentration of 30% (w / v). Reductive amination reaction was started by adding 0.5 M NaCNBH ₃ solution constituted in the reaction buffer to obtain a reducing agent having a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 5 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 14) and reverse phase chromatography on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the yield of conjugation. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

The HESylated EPO was isolated from unreacted compounds by cation exchange chromatography using an SP HP column on an Acta system (GE Healthcare). The eluent A was 20 mM sodium acetate, pH 4.0, and the eluent B was 20 mM sodium acetate, 1 M NaCl, pH 4.0. The gradient for separating the conjugate from unmodified protein was 0% B => 52% B in 13 CV (FIG. 15).

Example 12: Preparation of HES100 / 1.0 granulocyte colony stimulating factor (G-CSF) conjugate from HES

The deprotected HES aldehyde from Example 9 was dissolved in rh-Met-G-CSF solution [reaction buffer 5 mg / ml in 0.1 M sodium acetate buffer, pH 5; It has nearly the same amino acid sequence as the commercially available Neupogen® (supplier: Amgen, Munchen, D). G-CSF expressed by cola]. HES aldehyde was added at a 40-fold molar excess (based on M _n ) compared to the G-CSF concentration. The G-CSF concentration in the resulting reaction mixture was 1.3 mg / ml and the HES aldehyde concentration was 20% (w / v). Reductive amination reaction was started by adding 0.5 M NaCNBH ₃ solution constituted in the reaction buffer to obtain a reducing agent having a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 10 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE and reverse phase chromatography on a C18 column (Phenomenex, Jupiter) to determine the yield of conjugation. Elution of the RP-HPLC was performed using an acid water / acetonitrile gradient with 0.1% TFA.

The reaction mixture was analyzed by SDS-PAGE (Figure 16) and reverse phase chromatography (Figure 17) on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the conjugation yield. Elution of the RP-HPLC was performed using an acid water / acetonitrile gradient with 0.1% TFA.

The HESylated G-CSF was isolated from unreacted compounds by cation exchange chromatography using an SP HP column on an Acta system (GE Healthcare). The eluent A was 20 mM sodium acetate, pH 4.0, and the eluent B was 20 mM sodium acetate, 1 M NaCl, pH 4.0. The gradient for separating the conjugate from unmodified protein was 0% B => 40% B in 15 CV (FIG. 18).

Example 13: Pharmacodynamic in vivo bioassay in mice (HES-EPO conjugate according to Example 11)

A Balb C mouse weighing approximately 18 to 20 grams (purchased from Harlan Winkelmann GmbH, Borchen, Germany) was weighed in a Euro Standard Typ III (LxBxH 425x266x185 mm) at room temperature and relative humidity of 55% (Up to 10 birds per one). "Tapvei Einstreu" &lt; / RTI &gt; 4x4x1 mm (aspen) was used as a bedding material for our use. In addition, wood shavings were provided. We changed and cleaned us once a week. Drinking water (pH 3.8-4; sulfuric acid) was supplied unlimited. The animals numbered us. The animals within us were labeled in the ear and additionally labeled.

An initial physical examination was performed on the day of the approximate allocation and one week before starting treatment. Only healthy animals were used.

The HES EPO conjugate, unmodified starting material (rHuEPO) and Aranesp® (supplied by Amgen) as obtained in Example 11 were mixed at a dose of 100 μg / kg body weight, based on the protein content of the sample, 4 &lt; / RTI &gt; mice were tested as single swallowing subcutaneous dose. Equal volumes of PBS were included as vehicle controls.

Approximately 30 to 60 μl of whole blood samples at various time points (0, 3, 6, 9, 13, 16, 20 and 23 days) were incubated in a "Hamatokrit-Kapillaren" containing Na- The whole blood was centrifuged at 10,000 rpm for 6 minutes in a Hettich Hamatocrit 210 centrifuge (Tuttlingen, Germany), and the whole blood was collected by centrifugation The hematocrit ratio of the sample was determined. Erythropoietic response and duration were monitored as a function of time as a function of percent change in hematocrit (see FIG. 19).

These data show that hematocrit could be elevated in all samples containing EPO, Aranesp® or EPO conjugates. Aranesp® was able to increase its potency by 3 to 4 times compared to the starting material, and the HES EPO conjugate was able to increase its potency by 1.5 to 2 times compared to Aranesp®.

Example 14: Pharmacodynamic in vivo bioassay in mice (HES-IFN alpha conjugate according to Example 5)

One oxHES100 / 1.0 interferon alpha conjugate prepared according to Example 5 was tested in an in vivo assay according to Example 13. The EC50 dilution of the serum samples was plotted anti-logarithmically against time after intravenous injection. The half-life was calculated from the slope of the exponential pit-curve. The half-life of the sample was 8.9 hours (see FIG. 20).

The relative in vitro activity of the oxHES100 / 1.0 interferon alpha conjugate prepared according to Example 5 compared to Intron A is shown in FIG. 21 (see Example 16, below, for determination of in vitro activity).

Example 15: Pharmacodynamic in vivo bioassay in mice (HES-IFN alpha conjugate according to Example 10)

Three HES100 / 1.0 interferon alpha conjugates prepared in accordance with Example 10 were tested in an in vivo assay according to Example 13. The median of the EC50 dilution of the serum sample was plotted anti-logarithmically against time after intravenous injection. The half-life was calculated from the slope of the exponential pit-curve. The average half-life of the sample was 9.7 hours (see FIG. 22).

In the case of unmodified IFN-alpha, the serum antiviral activity was too low to calculate the serum half-life. K.R. Reddy et al. Advaced Drug Delivery Reviews 54 (2002) pp. 571-586] determined serum half-life (2 hours) of IFN-alpha in rats (intravenous).

The relative in vitro activity of three HES100 / 1.0 interferon alpha conjugates prepared according to Example 10 compared to Intron A is shown in FIG. 23 (see Example 16, below, for determination of in vitro activity).

Example  16: Explanation of the test procedure: Interferon-alpha ( Example  14 and 15)

After serial pre-dilution of the test items in the cell culture medium, a series of 2-fold dilutions were prepared. In a 96-well microtiter plate, diluted interferon (4x duplicates per dilution) was added to freshly trypsinized MDBK cells (40,000 cells per well). The assay was incubated at 37 占 폚 for 24 hours (total volume per well: 175 占 퐇).

Subsequently, a 50 μl diluted VSV stock solution was added to each well (except for positive control wells), resulting in a multiplicity of infection of 0.1.

The following controls were included in each assay: Twelve wells ( negative control ) with virus added to cell culture medium in place of interferon and 12 wells ( positive control ) with cell culture medium added instead of interferon and virus. The assay was incubated at 37 DEG C for 42 hours.

At the end of the incubation period, the cell culture supernatant from each well was replaced with 50 μl of MTT solution (at least 2 mg / mL in cell culture medium). The cells were incubated for 3 hours. The purple Formazan dye formed by proliferating cells was solubilized by adding 100 [mu] l of a solution of isopropanol / HCl (isopropanol with 40 mM HCl) to each well. Subsequently, the absorbance values of these solutions were measured at 570/630 nm on a microtiter plate reader.

The proliferative activity of MDBK cells grown in the presence of interferon and VSV was calculated for each dilution of interferon as follows:

(Mean absorbance of 4 interferon treated wells) - (mean absorbance of negative control)) * 100

-------------------------------------------------- -------------------

(Mean absorbance of positive control group) - (average absorbance of negative control group)

The antiviral activity of interferon-alpha was determined in four separate assays for each test item.

In the above-mentioned assay system, each conjugated HES100 / 1.0 interferon alpha conjugate (Examples 15 and 10 respectively) and oxHES100 / 1.0-interferon alpha conjugate (Examples 14 and 5 respectively) I. E. Intron A &lt; / RTI &gt; The CPE50 concentration of the material was calculated.

Example 17: Preparation of HES-Linker Derivative According to the Invention

In Example 17, the HES-linker derivative of the present invention was prepared. On the other hand, for a given linker structure, HES was varied in terms of average molecular weight and molar substitution thereof. On the other hand, for certain HES starting materials, the chemical properties of the linkers were varied.

The HES in the amounts shown in the following Tables 1 and 2 were dissolved in a sodium acetate buffer (1 mol / l, pH = 5) in a suitable volume ("Buffer V") by vigorously stirring and heating . To the clean solution, the indicated amount of linker (40 equivalents related to M _n of HES species) was added. In some cases, this amount of linker was added as a "DMF-linker" solution. Thus, the required amount of linker was dissolved in a small amount of DMF and the resulting clean DMF-Linker solution (labeled as "DMF-Linker solution V" in Table 2) was added to the reaction mixture. Finally, the solid NaCNBH ₃ , expressed as the NaCNBH ₃ amount, was dissolved in the stirred solution to give a final concentration of typically 0.6 M, and the reaction mixture was heated and stirred at 60 ° C for 18 to 24 hours.

To post-treat the reaction, the mixture was diluted with ultrapure water to give a final concentration of about 100 mg / ml (10% m / v) of the HES derivative, using ultrahigh purity as the solvent, kDa membrane or by ultrafiltration (UF). In case of linkers (a2) and (a3), 10 mM NH ₄ HCO ₃ - buffer, pH = 9 was used for ultrafiltration before using ultra high purity.

For subsequent deprotection, the purified HES-derivative solution (10%, 100 mg / ml) was acidified with a concentrated HCl solution to yield "c (HCl)" with an appropriate "pH level". The mixture is post-treated by ultrafiltration (membrane cut-off 10 kDa) using a suitable "post-treatment solvent" after neutralization (dilute NaOH) with stirring and heating at 40 캜 for reaction time "t" To give a white to yellow powder.

The derivatization was confirmed by successful coupling with the target molecule (Chemtid; see Example 18, Tables 3, 4 and 5 below). In the case of HES-derivatives prepared using linkers (a15) and (a10), successful derivatization was examined by spectroscopic properties. All HES-derivatives prepared as mentioned above were used for conjugation with the targets listed in Example 18, Tables 3, 4 and 5.

Abbreviations used in Tables 1 and 2:

D: Dialysis

DMF: Dimethylformamide

HES: hydroxyethyl starch

HCl: hydrochloric acid

NaCNBH ₃ : Sodium cyanoborohydride

NaOH: Natrium hydroxide

UF: Ultrafiltration

V: volume

Water: ultra high purity water (milliQ).

HES derivatives of HES 100 / 1.0 and linker structure (a2) were prepared according to Examples 8 and 9 of the present invention. The linker structure (a2) relates to structure (a2) as defined above, i.e. to the following 1-amino-3,3-diethoxypropane:

[Table 1]

Changes in the HES moiety (Example 17)

Changes in the HES portion (Example 17 continued)

* NH ₄ HCO ₃ - buffer (10 mM, pH = 9) was used for ultrafiltration before ultra pure water was used.

[Table 2]

Change of linker structure (Example 17)

Change of linker structure (Example 17 continued)

# As defined in the context of the present invention.

* DMSO instead of sodium acetate buffer.

** Centrifuge the diluted and neutralized reaction mixture before ultrafiltration.

And 40 were used instead of the 20 equivalents (related to the HES M _n).

Example 18: Preparation of HES-Linker-Biologically Active Derivatives According to the Invention

Positive target molecules as indicated in the following Tables 3, 4 and 5 were transferred into the appropriate reaction buffer. The indicated amount of HES-linker derivative (defined by linker and HES species) was dissolved in the reaction buffer and mixed with the target material solution. NaCNBH ₃ - typically present as a freshly prepared 0.5 M stock solution in reaction buffer - was added to a final concentration of typically 20 mM. The reaction mixture was incubated at temperature "rxnT" for a reaction time "rxn t " under temperature control.

The final reaction volume ("rxn V") and the concentration and ratio of the resulting reactant are given in Tables 3, 4 and 5.

The success of the conjugation reaction was confirmed by chromatographic analysis (RP-HPLC, SE-HPLC) or SDS-PAGE (see Figures 28-33 for the selected derivatives). In all coupling reactions described herein, a target-HES conjugate was detectable. The reaction conditions for various target molecules were not optimized.

Abbreviations Used:

rhIFNα: Recombinant human interferon-alpha 2b

rhEPO: recombinant human erythropoietin

rhG-CSF: Recombinant human granulocyte colony stimulating factor with additional N-terminal methionine

rhFIX: Recombinant human coagulation factor IX

rhFVIIa: Recombinant human coagulation factor VIIa

rhGH: recombinant human growth hormone

hFab; Fab fragments derived from human immunoglobulin G molecules

mIgG: Murine immunoglobulin G

GLP-1: glucagon-like peptide-1; Amino acid 1-37

r Asparaginase: This. Recombinant asparaginase from E. coli

NH ₂ -DNA: Sequence GGC TAC GTC CAG GAG CCA Oligonucleotides with a 5'-aminohexyl spacer with CCT

rh-leptin: Recombinant human leptin

Ampou B: Amphotericin B, CAS No. 1397-89-3

Kemp lactide: Trp ⁴ - Kemp lactide (Leu-Arg-Arg-Trp -Ser-Leu-Gly), CAS No. 80224-16-4

NaOAc: Buffer containing sodium acetate

HEPES: 4- (2-hydroxyethyl) -1-piperazinethanesulfonic acid, CAS No. &lt; / RTI &gt; 7365-45-9

[Table 3]

Change of target substance (Example 18)

Change of target substance (Example 18 continued)

[Table 4]

Changes in the HES moiety (Example 18)

Changes in the HES portion (Example 18 continued)

[Table 5]

Change of linker structure (Example 18)

Changes in linker structure (Example 18 continued)

Additional data

(A.1) HBS  7 kDa from oxHBS -N- (3- Propioldehyde diethyl acetal )

Starch (HBS) aldonic acid was synthesized starting from highly branched starch [M _w = 7000 daltons (7 kDa), average degree of branching: 15 mol%] according to Example 9 of WO2005 / 083103 A1. The aldonic acid so obtained was transferred to the corresponding lactone by drying at 80 DEG C for 24 hours (the abbreviation "oxHBS " refers to HBS aldonic acid as well as the corresponding lactone).

5 g of lactone was dissolved in 15 ml of 1-amino-3,3-diethoxypropane and 10 ml of anhydrous DMF and stirred at 70 DEG C for 48 hours. Excess 1-amino-3,3-diethoxypropane and DMF (dimethylformamide) were evaporated in vacuo and the resulting pale yellow solid was washed with acetone until the yellow color disappeared. The product was dissolved in water and purified by ultrafiltration using a membrane with a cut-off of 1,000 daltons until the pH of the filtrate reached a value of > 6.

The preservate was treated with 2 g of acidic cation exchange resin (Amberlite (R) 120) for 2 hours, the resin was filtered off and the remaining solution was lyophilized.

The &lt; ¹ &gt; H-NMR spectrum of the compound showed a triplet at 1.7 ppm and a polyline at 1.2 ppm, indicating that the residue of the linker compound (1-amino-3,3-diethoxypropane) A methyl group and a methylene group.

(A.2) Preparation of oxHBS 7 kDa-Bovine Serum Albumin (BSA) conjugate

750 μg of the acetal prepared in (A.1) was dissolved in 5 ml of 0.01 N HCl. The pH was adjusted to 2.0 with 1 N HCl and the reaction mixture was stirred at 21 &lt; 0 &gt; C for 18 hours. 2 ml of 1% BSA solution in acetone buffer (pH = 7.0) was added to 200 [mu] l of the prepared mixture. 140 mg of sodium cyanoborohydride was dissolved in 5 ml of 0.1 N acetate buffer (pH = 7.0), and 50 ml aliquots were immediately added to the reaction mixture. The reaction mixture was stored at 4 &lt; 0 &gt; C for 15 hours. The reaction mixture was analyzed by size exclusion chromatography and the reaction yield of the HBS-BSA conjugate was found to be 90% (FIG. 24).

(A.3) Preparation of oxHBS 65 kDa-interferon-alpha conjugate

(W / v) was added to 400 mg of 65 kDa HBS-N- (3-propioldehyde diethyl acetal) prepared in analogy to (A.1) This two solution was calculated. The solution was incubated overnight at &lt; RTI ID = 0.0 &gt; 21 C &lt; / RTI &gt; with stirring to deprotect the aldehyde functionality. The pH value was adjusted to the value used in the conjugate buffer by adding 0.1 M NaOH prior to coupling.

Recombinant human interferon alpha-2b produced by recombinant DNA technology using interferon-alpha [Escherichia coli (E. coli)]; Such interferon alpha-2b is composed of 165 amino acids and presents the same amino acid sequence as natural human interferon alpha-2b (hIFN-alpha-2b)] is concentrated to 16 mg / ml or less, And transferred to a suitable conjugation buffer (0.1 M sodium acetate buffer, pH 4.0).

A 10-fold molar excess of oxHBS aldehyde (based on M _w ) was used with a final protein concentration of 6 mg / ml in the reaction mixture; The concentration of oxHBS aldehyde was 20% (w / v). The deprotected oxHBS aldehyde was combined with the protein solution and a reductive amination reaction was initiated by adding freshly prepared NaCNBH ₃ solution (0.5 M in conjugation buffer) to obtain a reducing agent with a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 10 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 25) and reverse phase chromatography on a C18 column (Phenomenex, Jupiter) to demonstrate successful coupling and determine the yield of conjugation. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

The HBS-interferon-alpha was isolated from unreacted compounds by anion exchange chromatography using a Q HP column on an Akta system (GE Healthcare). The eluent A was 10 mM Tris-Cl, pH 8.0, and the eluent B was 10 mM Tris-Cl, 0.5 M NaCl, pH 8.0. The gradient for separating the conjugate from the unmodified protein was 0% B => 50% B in 16 CV (FIG. 26).

(A.4) Preparation of oxHBS 65 erythropoietin (EPO) conjugate

(W / v) was added to 400 mg of 65 kDa HBS-N- (3-propioldehyde diethyl acetal) prepared in analogy to (A.1) with 10 mM HCl in an appropriate amount Respectively. The solution was incubated overnight at &lt; RTI ID = 0.0 &gt; 21 C &lt; / RTI &gt; with stirring to deprotect the aldehyde functionality. The pH value was adjusted to the value used in the conjugation buffer by adding 0.1 M NaOH.

The deprotected oxHBS aldehyde was dissolved in EPO [recombinant human EPO] solution having amino acid sequence of human EPO and having almost the same characteristics as commercially available Erypo® (Ortho Biotech, Jansen-Cilag) or NeoRecormon® (supplied by Roche) 10 mg / ml in reaction buffer, 0.1 M sodium acetate buffer, pH 5). The OxHBS aldehyde was added in a 20-fold molar excess (based on M _w ) compared to the EPO concentration. The resulting reaction mixture had an EPO concentration of 4.6 mg / ml and an oxHBS aldehyde concentration of 20% (w / v). Reductive amination reaction was started by adding 0.5 M NaCNBH ₃ solution constituted in the reaction buffer to obtain a reducing agent having a final concentration of 20 mM. After thorough mixing, the reaction was incubated overnight at &lt; RTI ID = 0.0 &gt; 10 C. &lt; / RTI &gt;

The reaction mixture was analyzed by SDS-PAGE (Figure 27) and by reverse phase chromatography on a C18 column (Phenomenex, Jupiter) (Figure A.4-1) to demonstrate successful coupling and determine the conjugation yield. Elution was carried out using an acid water / acetonitrile gradient with 0.1% TFA.

Claims (54)

(i) reacting a hydroxyalkyl starch (HAS) of formula (I) with an amino group M of a cross-linking compound according to formula II via a carbon atom C * at the reducing end of the HAS, Step of obtaining a derivative
A method for producing a hydroxyalkyl starch derivative, comprising:
Formula I

(II)
MLA
(III)

In this formula,
C * is optionally oxidized prior to reaction with HAS and M;
X is a functional group generated by reacting the amino group M with HAS through the optionally oxidized reducing end carbon atom C * of HAS;
HAS 'is the remainder of the hydroxyalkyl starch molecule; R ₁ , R ₂ and R ₃ are independently hydrogen or a linear or branched hydroxyalkyl group;
A is a residue according to the following formula (IIa)
&Lt; RTI ID =

[In the above formula,
Z ₁ and Z ₂ are each independently O or S,
A ₁ and A ₂ each independently represent a methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl or a ring according to the formula:
&Lt; RTI ID =

(Wherein A ₁ and A ₂ together are - (CH ₂ ) ₂ - or - (CH ₂ ) ₃ - or - (CH ₂ CH (CH ₃ )
A ₃ is H or together with the N atom of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl or amino group M or together with appropriate atoms contained in L form a ring;
Amino group M is a group according to the following formula &lt; RTI ID = 0.0 &gt; (IIc)
&Lt; RTI ID =

Wherein Y is absent or

(Wherein G is O or S or NH)
&Lt; / RTI &gt; is a chemical moiety selected from the group consisting of &
R 'is H or an organic moiety selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and tertiary butyl;
L is - (CL ₁ L ₂ ) _n -, wherein n is an integer from 1 to 20, or
L is - ((CL ₁ L ₂ ) ₂ -O) _m - (CL ₁ L ₂ ) - wherein m is 1, 2 or 3, or
L is - (CL ₁ L ₂ ) _n - (C═O) -NH- (CL ₁ L ₂ ) _n - wherein each n independently ranges from 1 to 4,
L ₁ and L ₂ is independently of each other H or an organic moiety selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and tertiary butyl optionally substituted with fluoro, chloro or bromo. The method of claim ₁ , wherein R ₁ , R ₂ and R ₃ are independently a group - (CH ₂ CH ₂ O) _n -H and n is 0, 1, 2, 3, 4, 5 or 6. 3. The method of claim 1 or 2, wherein the hydroxyalkyl starch is a hydroxyethyl starch (HES). 2. The method of claim 1, wherein A &lt; ₃ &gt; The method of claim 1, wherein Z ₁ and Z ₂ are each O, A ₃ and is H, A ₁ and A ₂ are each ethyl, or or A ₁ and A ₂ is unsubstituted (wherein, A ₁ and A according to the formula IIb ₂ together form - (CH ₂ ) ₂ -. delete The method according to claim 1, wherein the amino group M is H ₂ N-, H ₂ NO-, H ₂ N-NH- (C═O) -, H ₃ C-NH- or H ₃ C-NH-O- . The method of claim 1 wherein in (i) the HAS is reacted with its amino group M of the crosslinking compound via its oxidized reducing end, wherein M is H ₂ N-, (Wherein X is - (C = O) -NH-). The method according to claim 1, wherein in (i), the HAS is reacted with its amino group M (where M is H ₂ N-) through its non-oxidized reducing end in an aqueous system, Wherein the reaction is carried out at a temperature in the range of from 20 to 80 C at a pH in the range of from 4 to 7, wherein X is -CH = N-. 10. The method of claim 9, HAS derivative to carry out the reaction in (i), in the presence of a reducing agent method for obtaining a (where, X is -CH ₂ -NH-). The method of claim 1, wherein in (i) the HAS is reacted with an amino group M of the crosslinking compound, wherein M is H ₂ NO- or H ₂ N-NH- (C = O) - and the reaction is carried out at a temperature in the range of from 5 to 80 ° C at a pH in the range of from 4.5 to 6.5, wherein X is -CH = NO- or -CH = N-NH- (C = O) -. 2. The compound according to claim 1, wherein L &lt; ₁ & L &lt; ₂ &gt; is H. 2. The method of claim 1, wherein L is - (CL ₁ L ₂ ) _n - and n is an integer from 1 to 10. The compound of claim 13, wherein L is - (CH ₂ ) _n -, - ((CH ₂ ) ₂ -O) _m -CH ₂ -, or - (CH ₂ ) _n - ₂ ) _n -. 15. The method of claim 14, L is -CH ₂ -CH ₂ - Method a. delete 15. The compound of claim 14, wherein L is - (CH ₂ ) ₃ - (C = O) -NH- (CH ₂ ) ₃ -; - (CH ₂ ) ₃ - (C = O) -NH- (CH ₂ ) ₂ -; - (CH ₂ ) ₂ - (C = O) -NH- (CH ₂ ) ₃ -; And - (CH ₂ ) ₂ - (C = O) -NH- (CH ₂ ) ₂ -. delete delete The method according to claim 1,
(ii) reducing amination of the HAS derivative according to formula III with an amino group of the biologically active agent H ₂ N-BA 'through group A to give a HAS derivative according to formula IV
Further comprising:
Formula IV

21. A method according to claim 20, wherein prior to step (ii), the group A of the HAS derivative according to formula (III) is converted to the corresponding aldehyde or keto group. The method according to claim 20 or 21, wherein in (ii) the reaction is carried out in an aqueous system in the presence of a reducing agent, at a temperature in the range of 0 to 37 ° C and at a pH in the range of 3 to 9, Wherein the molar ratio of the derivative to the biologically active agent BA is from 0.1: 1 to 200: 1 equivalents based on the number average molecular weight (M _n ) of the HAS derivative. 21. The method of claim 20, wherein the biologically active compound BA is selected from the group consisting of peptides, oligopeptides, polypeptides, proteins; Functional derivatives, fragments or analogues of such polypeptides or proteins; Small molecule compound or oligonucleotide. 24. The method of claim 23, wherein the protein is selected from the group consisting of a colony stimulating factor (CSF) such as erythropoietin (EPO) such as recombinant human EPO (rhEPO), G-CSF-like recombinant human G-CSF (rhG- (IFN) selected from the group consisting of IFN beta, IFN gamma-like recombinant human IFN alpha (rhIFN alpha) and recombinant human IFN beta (rhIFN beta), recombinant human factor VIIa (rhFVIIa), recombinant human factor IX such as Factor IX, growth hormone (GH) such as recombinant human growth hormone (rhGH), Fab fragments such as Fab fragments (hFab) derived from human immunoglobulin G molecules, murine immunoglobulin G (mIgG) Asparaginase such as an immunoglobulin G, a glucagon-like peptide-1 (GLP-1), a recombinant asparaginase (rasparaginase), leptin such as recombinant human leptin (rh leptin), interleukin- 11, alpha-1-antitrypsin, an antibody or The method of body segments, or alternative protein scaffold (scaffold). Hydroxyalkyl starch (HAS) derivatives of formula III:
(III)

In this formula,
X is selected from the group consisting of the amino group M of the crosslinking compound of the formula (II), the carbon atom C * of the hydroxyalkyl starch (HAS) of the following formula I, wherein C * is optionally oxidized before the reaction with HAS and M, Lt; RTI ID = 0.0 &gt; HAS &lt; / RTI &gt;
HAS 'is the remainder of the hydroxyalkyl starch molecule; R ₁ , R ₂ and R ₃ are independently hydrogen or a linear or branched hydroxyalkyl group;
Formula I

(II)
MLA
A is a residue according to the following formula (IIa)
&Lt; RTI ID =

[In the above formula,
Z ₁ and Z ₂ are each independently O or S,
A ₁ and A ₂ each independently represent a methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl or a ring according to the formula:
&Lt; RTI ID =

(Wherein A ₁ and A ₂ together are - (CH ₂ ) ₂ - or - (CH ₂ ) ₃ - or - (CH ₂ CH (CH ₃ )
A ₃ is H or together with the N atom of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl or amino group M or together with appropriate atoms contained in L form a ring;
Amino group M is a group according to the following formula &lt; RTI ID = 0.0 &gt; (IIc)
&Lt; RTI ID =

Wherein Y is absent or

(Wherein G is O or S or NH)
&Lt; / RTI &gt; is a chemical moiety selected from the group consisting of &
R 'is H or an organic moiety selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and tertiary butyl;
L is - (CL ₁ L ₂ ) _n -, wherein n is an integer from 1 to 20, or
L is - ((CL ₁ L ₂ ) ₂ -O) _m - (CL ₁ L ₂ ) - wherein m is 1, 2 or 3, or
L is - (CL ₁ L ₂ ) _n - (C═O) -NH- (CL ₁ L ₂ ) _n - wherein each n independently ranges from 1 to 4,
L ₁ and L ₂ is independently of each other H or an organic moiety selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and tertiary butyl optionally substituted with fluoro, chloro or bromo. A compound according to claim 25, wherein R ₁ , R ₂ and R ₃ are independently a group - (CH ₂ CH ₂ O) _n -H and n is 0, 1, 2, 3, 4, 5 or 6 . 26. The HAS derivative according to claim 25 or 26, wherein the hydroxyalkyl starch is hydroxyethyl starch (HES). 26. A HAS derivative according to claim 25, wherein A &lt; ₃ &gt; is H: A compound according to claim 25, wherein X is -CH = N-, -CH ₂ -NH-, -CH = NO-, -CH ₂ -NH-O-, -C (= 0) -NH-NH- and -CH = N-NH- (C = O) -. delete 26. The compound of claim 25, wherein L &lt; _{1 &gt;} and A HAS derivative wherein L &lt; ₂ &gt; 26. The method of claim 25, L is _{- (CL 1 L 2) n} - wherein n is HAS derivative is 1 to 10. 26. The method of claim 25, L is _{- (CH 2) n -,} - ((CH 2) 2 -O) m -CH 2 -, - (CH 2) n - (C = O) -NH- (CH 2 ) _n -. 34. The method of claim 33, L is -CH ₂ -CH ₂ - the HAS derivative. 34. The method of claim 33, L is _{- (CH 2) 3 - (} C = O) -NH- (CH 2) 3 -; - (CH ₂ ) ₃ - (C = O) -NH- (CH ₂ ) ₂ -; - (CH ₂ ) ₂ - (C = O) -NH- (CH ₂ ) ₃ -; And - (CH ₂ ) ₂ - (C = O) -NH- (CH ₂ ) ₂ -. The hydroxyalkyl starch derivative according to claim 25 having the structure:

27. The hydroxyalkyl starch derivative according to claim 26 having the structure:

Hydroxyalkyl starch derivatives of formula IV:
Formula IV

Wherein X is selected from the group consisting of the amino group M of the following cross-linking compound of the formula (II) with the carbon atom C * of the hydroxyalkyl starch (HAS) of the following formula I, Wherein X is not an amide group -C (= O) -NH-) via reaction with a HAS of formula (I)
Formula I

(II)
MLA
Wherein HAS 'is the remainder of the hydroxyalkyl starch molecule; R ₁ , R ₂ and R ₃ are independently hydrogen or a linear or branched hydroxyalkyl group;
A is a residue according to the formula &lt; RTI ID = 0.0 &gt;
&Lt; RTI ID =

[In the above formula,
Z ₁ and Z ₂ are each independently O or S;
A ₁ and A ₂ are each independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, tert- butyl, benzyl,
&Lt; RTI ID =

Wherein A ₁ and A ₂ together are - (CH ₂ ) ₂ - or - (CH ₂ ) ₃ - or - (CH ₂ CH (CH ₃ )) -, A ₃ is H or methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, benzyl, or together with the N atom of the amino group M or together with the appropriate atoms contained in L, form a ring;
Amino group M is a group according to the following formula &lt; RTI ID = 0.0 &gt; (IIc)
&Lt; RTI ID =

Wherein Y is absent or

(Wherein G is O or S or NH)
&Lt; / RTI &gt; is a chemical moiety selected from the group consisting of &
R 'is H or an organic moiety selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and tertiary butyl;
L is - (CL ₁ L ₂ ) _n -, wherein n is an integer from 1 to 20, or
L is - ((CL ₁ L ₂ ) ₂ -O) _m - (CL ₁ L ₂ ) - wherein m is 1, 2 or 3, or
L is - (CL ₁ L ₂ ) _n - (C═O) -NH- (CL ₁ L ₂ ) _n - wherein each n independently ranges from 1 to 4,
L ₁ and L ₂ is independently from each other H or an organic residue selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl and tertiary butyl optionally substituted with fluoro,
BA 'is the remainder of the amino group of BA via reductive O folk reaction by reacting with A, or remaining after the aldehyde group or keto group corresponding to a reaction A, a biologically active agent BA'-NH _2. 39. The method of claim 38, HAS derivative is selected from the group consisting of X is _{-CH 2 -NH-, -CH = N-,} -CH 2 -NH-O- and -CH = NO-. 39. The HAS derivative of claim 38, wherein A &lt; ₃ &gt; 39. The compound of claim 38, wherein L &lt; _{1 &gt;} and A HAS derivative wherein L &lt; ₂ &gt; 39. The HAS derivative of claim 38, wherein L is - (CL ₁ L ₂ ) _n - and n is 1 to 10. 39. The method of claim 38, L is _{- (CH 2) n -,} - ((CH 2) 2 -O) m -CH 2 -, - (CH 2) n - (C = O) -NH- (CH 2 ) _n -. 34. The method of claim 33, L is -CH ₂ -CH ₂ - the HAS derivative. 45. The method of claim 44, L is _{- (CH 2) 3 - (} C = O) -NH- (CH 2) 3 -; - (CH ₂ ) ₃ - (C = O) -NH- (CH ₂ ) ₂ -; - (CH ₂ ) ₂ - (C = O) -NH- (CH ₂ ) ₃ -; And - (CH ₂ ) ₂ - (C = O) -NH- (CH ₂ ) ₂ -. 39. The HAS derivative of claim 38 having the structure:

39. The method of claim 38, wherein the biologically active compound BA is selected from the group consisting of peptides, oligopeptides, polypeptides, proteins; Functional derivatives, fragments or analogues of such polypeptides or proteins; HAS derivatives which are small molecule compounds or oligonucleotides. 48. The method of claim 47, wherein the protein is selected from the group consisting of a colony stimulating factor (CSF) such as erythropoietin (EPO) such as recombinant human EPO (rhEPO), G-CSF- like recombinant human G- (IFN) selected from the group consisting of IFN beta, IFN gamma-like recombinant human IFN alpha (rhIFN alpha) and recombinant human IFN beta (rhIFN beta), recombinant human factor VIIa (rhFVIIa), recombinant human factor IX such as Factor IX, growth hormone (GH) such as recombinant human growth hormone (rhGH), Fab fragments such as Fab fragments (hFab) derived from human immunoglobulin G molecules, murine immunoglobulin G (mIgG) Asparaginase such as an immunoglobulin G, a glucagon-like peptide-1 (GLP-1), a recombinant asparaginase (rasparaginase), leptin such as recombinant human leptin (rh leptin), interleukin- 11, alpha-1-antitrypsin, an antibody or Body fragments, or alternative protein scaffold (scaffold) of HAS derivative. 39. The HAS derivative according to claim 38, as a therapeutic or prophylactic agent. 39. The HAS derivative of claim 38 for use in a method of treatment of human or animal. 38. A pharmaceutical composition comprising a therapeutically effective amount of a HAS derivative according to claim 38. The method of claim 1, wherein the crosslinking compound MLA according to formula (II) is selected from the group consisting of compounds of the formula:

53. The method of claim 52, wherein the crosslinking compound MLA according to formula &lt; RTI ID = 0.0 &gt;

&Lt; / RTI &gt; 54. The method of claim 53, wherein the crosslinking compound MLA according to formula (II) is 1-amino-3, 3-diethoxypropane.

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